Integrated analytical system and method

ABSTRACT

An analytical assembly within a unified device structure for integration into an analytical system. The analytical assembly is scalable and includes a plurality of analytical devices, each of which includes a reaction cell, an optical sensor, and at least one optical element positioned in optical communication with both the reaction cell and the sensor and which delivers optical signals from the cell to the sensor. Additional elements are optionally integrated into the analytical assembly. Methods for forming and operating the analytical system are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/786,215 filed Oct. 17, 2017 which is a continuation of U.S. patentapplication Ser. No. 15/272,138 filed Sep. 21, 2016 which is acontinuation of U.S. patent application Ser. No. 14/730,970 filed Jun.4, 2015 which is a continuation of U.S. patent application Ser. No.14/477,323 filed Sep. 4, 2014 (now U.S. Pat. No. 9,291,568) which is acontinuation of U.S. patent application Ser. No. 14/107,888 filed Dec.16, 2013 (now U.S. Pat. No. 8,867,038) which is a continuation of U.S.patent application Ser. No. 13/895,629 filed May 16, 2013 (now U.S. Pat.No. 8,649,011) which is a continuation of U.S. patent application Ser.No. 13/031,122 filed Feb. 18, 2011 (now U.S. Pat. No. 8,467,061) whichclaims priority to U.S. Provisional Application No. 61/306,235 filedFeb. 19, 2010, U.S. Provisional Patent Application No. 61/410,189 filedNov. 4, 2010 and U.S. Provisional Patent Application No. 61/387,916filed Sep. 29, 2010, the entire contents of which applications isincorporated herein for all purposes by this reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not Applicable.

BACKGROUND OF THE INVENTION

Analytical technologies continue to advance far beyond the test tubescale evaluations of the 19th and 20th centuries, and have progressed tothe point where researchers can look at very specific interactions invivo, in vitro, at the cellular level, and even at the level ofindividual molecules. This progression is driven not just by the desireto understand important reactions in their purest form, but also by therealization that seemingly minor or insignificant reactions in livingsystems can prompt a cascade of other events that could potentiallyunleash a life or death result.

In this progression, these analyses not only have become more focused onlesser events, but also have had to become appropriately more sensitive,in order to be able to monitor such reactions. In increasing sensitivityto the levels of cellular or even single molecular levels, one mayinherently increase the sensitivity of the system to other non-relevantsignals, or ‘noise’. In some cases, the noise level can be of sufficientmagnitude that it partially or completely obscures the desired signals,i.e., those corresponding to the analysis of interest. Accordingly, itis desirable to be able to increase sensitivity of detection whilemaintaining the signal-to-noise ratio.

There is a continuing need to increase the performance of analyticalsystems and reduce the cost associated with manufacturing and using thesystem. In particular, there is a continuing need to increase thethroughput of analytical systems. There is a continuing need to reducethe size and complexity of analytical system. There is a continuing needfor analytical systems that have flexible configurations and are easilyscalable.

The present invention provides devices, systems and methods forovercoming the above problems in addition to other benefits.

BRIEF SUMMARY OF THE INVENTION

The present invention is generally directed to an integrated analyticaldevice that includes, within a single unified structure, a plurality ofreaction cells, at least one detector element, and an optical elementfor delivering an optical signal from a respective reaction cell to thedetector element. A variety of elements may be integrated into thedevice structure that enhances the performance and scalability of thedevice. Various aspects of the invention are directed to an analyticalsystem employing an integrated analytical device and elements andmethods for efficient integration.

The methods and apparatuses of the present invention have other featuresand advantages which will be apparent from or are set forth in moredetail in the accompanying drawings, which are incorporated herein, andthe following Detailed Description of the Invention, which togetherserve to explain certain principles of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a block diagram of an optical analytical device architecturein accordance with the present invention. FIG. 1B schematicallyillustrates an end view of an integrated optical analysis device,including waveguide illumination. FIG. 1C illustrates the majorcomponents of the integrated device illustrated in FIG. 1B, from a topview.

FIG. 2A and FIG. 2B are schematic illustrations of an integratedanalytical device (optode) of the invention.

FIG. 3 is a schematic illustration of integration of an integratedanalytical device (optode) into an optode array chip in accordance withthe present invention.

FIG. 4 is a schematic view of a topside connection of fluidic andillumination elements to an integrated analytical device.

FIG. 5A is a schematic view a test socket and analytical system. FIG. 5Bis a perspective view of an exemplary test socket receiving an exemplaryanalytical optode chip.

FIG. 6 is a schematic view of a micro-pipette array for deliveringreagent to an optode chip array.

FIG. 7 is a cross-sectional view of the test socket and analyticalsystem illustrating microfluidic connections.

FIG. 8 is a schematic view of a top portion of the test socketillustrating distributed photonics and fluidics system.

FIG. 9 is a top view of a test socket of FIG. 8.

FIG. 10 is a schematic view of an array of reaction cells in atransparent substrate, illustrating an embodiment for providingillumination light to the reaction cells.

FIG. 11 is an exemplary plot of interleaved electrical data connectionsversus single molecule waveguides.

FIG. 12 is a schematic view of an analytical device having directionalcomponents defining an optical guide path, the device being formed fromlayers on a substrate.

FIG. 13A-FIG. 13E shows various layers in an optode array chip. FIG. 13Ais a layer having detectors and processing components, FIG. 13B is a topview of the device showing distributed fluidics and illuminationsystems. FIG. 13C shows the bottom of the device having electricalcontacts for connections with distributed power and signal systems.FIGS. 13D and 13E schematically illustrate a hybrid fabrication joiningprocess. FIG. 13D schematically illustrates the use of ahydrophobic/hydrophilic surface interaction in joining and aligningcomponent substrates. FIG. 13E schematically illustrates the processintroducing spacing elements between the substrates.

FIG. 14 shows views of various configurations for arrangements of optodearray components, fluidic input components, and illumination inputcomponents.

FIG. 15 is a schematic view of an array of reaction cells and theoptical emission profiles emanating from those reaction cells.

FIG. 16. is a schematic view of an optical containment structure for usein accordance with the invention.

FIG. 17 is a cross-sectional, schematic view of pixels of a typical CMOSsensor used in the system in accordance with the present invention.

FIG. 18 schematically illustrates one structure of an opticalcontainment structure of the invention.

FIG. 19 schematically illustrates one process flow for fabrication ofthe structure shown in FIG. 18.

FIG. 20 is a schematic view of an alternate optical containmentstructure of the invention employing a mix of diffractive and reflectivematerials.

FIG. 21A, FIG. 21B and FIG. 21C show exemplary plots of interleavedexcitation illumination and signal data using a system similar to thatof FIG. 23

FIG. 22 is a schematic view of a pixel design with optionally gatedstorage elements.

FIG. 23 is a schematic of an analytical system having a plurality ofintegrated analytical devices in accordance with the present invention.

FIG. 24 is a schematic of an analytical system having a plurality ofintegrated analytical devices.

FIG. 25 is a schematic diagram of an analytical device with an array ofreaction cells and waveguides configured for measuring scattering fromnanoparticles.

FIG. 26 is a schematic representation of a polling based approach todata synchronization in accordance with the present invention.

FIG. 27 is a schematic representation of an interrupt drivenarchitecture with reduced storage in an optode element in accordancewith the present invention.

FIG. 28 is a schematic representation of a smart pixel for event loggingwith multi-species discrimination in accordance with the presentinvention.

FIG. 29 is a schematic representation of differentiator circuit with aclamped capacitor in accordance with the present invention.

FIG. 30 is a schematic representation of a compact CMOS programmabletrigger circuit in accordance with the present invention.

FIG. 31 is a schematic representation of differential amplifier for atrigger circuit in accordance with the present invention.

FIG. 32 is a schematic representation of compact CMOS trigger circuit inaccordance with the present invention.

FIG. 33 is a schematic representation of two tap storage nodes from aphotodetector with non-destructive monitoring in accordance with thepresent invention.

FIG. 34 is a representative timing diagram of an event capture circuitsin accordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to the various embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings. While the invention will be described in conjunction with thevarious embodiments, it will be understood that they are not intended tolimit the invention to those embodiments. On the contrary, the inventionis intended to cover alternatives, modifications and equivalents, whichmay be included within the spirit and scope of the invention as definedby the appended claims.

I. Optical Analyses

The present invention is generally directed to improved systems, methodsand devices for use in optical analyses, and particularly, opticalanalyses of biological and/or chemical samples and reactions. Ingeneral, these optical analyses seek to gather and detect one or moreoptical signals, the appearance or disappearance of which, orlocalization of which, is indicative of a given chemical or biologicalreaction and/or the presence or absence of a given substance within asample material. In some cases, the reactants, their products, orsubstance of interest (all of which are referred to as reactants herein)inherently present an optically detectable signal which can be detected.In other cases, reactants are provided with exogenous labeling groups tofacilitate their detection. Useful labeling groups include fluorescentlabels, luminescent labels, mass labels, light scattering labels,electrochemical labels (e.g., carrying large charge groups), metallabels, and the like. Exemplars of such labeling groups are disclosed byU.S. Pat. No. 7,332,284 and U.S. Patent Publication Nos. 2009/0233302filed Mar. 12, 2009, 2008/0241866 filed Mar. 27, 2008, and 2010/0167299filed Nov. 17, 2009, the contents of which patents and applications areincorporated herein for all purposes by this reference.

In various embodiments, one or more reactants in an analysis is providedwith a fluorescent labeling group that possesses a fluorescent emissionspectrum that is shifted from its excitation spectrum, allowingdiscrimination between the excitation light source and the emission ofthe label group. These fluorescent labels typically have high quantumyields, further enhancing their detectability. A variety of differentfluorescent label groups are well known in the art, and includefluorescein and rhodamine based organic dyes, such as those sold underthe Cy3 and Cy5 labels from, e.g., GE Healthcare, and the AlexaFluor®dyes available from Life Technologies, Inc. A wide variety of organicdye structures have been previously described in the art.

Other fluorescent label groups include, for example, particle-basedlabeling groups. Some such particle label groups constitute encapsulatedor otherwise entrained organic fluorophores, while others comprisefluorescent nanoparticles, such as inorganic semiconductor nanocrystals,e.g., as described in U.S. Pat. Nos. 6,207,392, 6,225,198, 6,251,303,6,501,091, and 7,566,476, the full disclosures of which are incorporatedherein by reference in their entirety for all purposes.

By detecting these fluorescent labeling groups, one can ascertain thelocalization of a given labeled reactant, or detect reaction events thatresult in changes in the spectral or other aspects of the fluorescentlylabeled reactant. For example, in binding or hybridization reactions,the ability of a labeled reactant to bind to another immobilizedreactant is detected by contacting the reactants, washing unboundlabeled reactant away, and observing the immobilized reactant to lookfor the presence of bound fluorescent label. Such assays are routinelyemployed in hybridization assays, antibody assays, and a variety ofother analyses.

In a number of different nucleic acid sequencing analyses, fluorescentlylabeled nucleotides are used to monitor the polymerase-mediated,template-dependent incorporation of nucleotides in a primer extensionreaction. In particular, a labeled nucleotide is introduced to a primertemplate polymerase complex, and incorporation of the labeled nucleotideis detected. If a labeled nucleotide is incorporated, it is indicativeof the underlying and complementary nucleotide in the sequence of thetemplate molecule. In traditional Sanger sequencing processes, thedetection of incorporation of labeled nucleotides utilizes a terminationreaction where the labeled nucleotides carry a terminating group thatblocks further extension of the primer. By mixing the labeled terminatednucleotides with unlabeled native nucleotides, one generates nested setsof fragments that terminate at different nucleotides. These fragmentsare then separated by capillary electrophoresis, to separate thosefragments that differ by a single nucleotide, and the labels for thefragments are read in order of increasing fragment size to provide thesequence (as provided by the last added, labeled terminated nucleotide).By providing a different fluorescent label on each of the types ofnucleotides that are added, one can readily differentiate the differentnucleotides in the sequence (e.g., U.S. Pat. No. 5,821,058, incorporatedherein for all purposes by this reference).

In newer generation sequencing technologies, arrays of primer-templatecomplexes are immobilized on surfaces of substrates such that individualmolecules or individual and homogeneous groups of molecules arespatially discrete from other individual molecules or groups ofmolecules, respectively. Labeled nucleotides are added in a manner thatresults in a single nucleotide being added to each individual moleculeor group of molecules. Following the addition of the nucleotide, thelabeled addition is detected and identified.

In some cases, the processes utilize the addition of a single type ofnucleotide at a time, followed by a washing step. The labelednucleotides that are added are then detected, their labels removed, andthe process repeated with a different nucleotide type. Sequences ofindividual template sequences are determined by the order of appearanceof the labels at given locations on the substrate.

In other similar cases, the immobilized complexes are contacted with allfour types of labeled nucleotides where each type bears adistinguishable fluorescent label and a terminator group that preventsthe addition of more than one nucleotide in a given step. Following thesingle incorporation in each individual template sequence (or group oftemplate sequences,) the unbound nucleotides are washed away, and theimmobilized complexes are scanned to identify which nucleotide was addedat each location. Repeating the process yields sequence information ofeach of the template sequences. In other cases, more than four types oflabeled nucleotides are utilized.

In particularly elegant approaches, labeled nucleotides are detectedduring the incorporation process, in real time, by individual molecularcomplexes. Such methods are described, for example, in U.S. Pat. No.7,056,661, which is incorporated herein by reference in its entirety forall purposes. In these processes, nucleotides are labeled on a terminalphosphate group that is released during the incorporation process, so asto avoid accumulation of label on the extension product, and avoid anyneed for label removal processes that can be deleterious to thecomplexes. Primer/template polymerase complexes are observed during thepolymerization process, and nucleotides being added are detected byvirtue of their associated labels. In one particular aspect, they areobserved using an optically confined structure, such as a zero modewaveguide (See, e.g., U.S. Pat. No. 6,917,726, which is incorporatedherein by reference in its entirety for all purposes) that limitsexposure of the excitation radiation to the volume immediatelysurrounding an individual complex. As a result, only labeled nucleotidesthat are in the process of being incorporated are exposed to excitationillumination for a time that is sufficient to identify the nucleotide.In another approach, the label on the nucleotide is configured tointeract with a complementary group on or near the complex, e.g.,attached to the polymerase, where the interaction provides a uniquesignal. For example, a polymerase may be provided with a donorfluorophore that is excited at a first wavelength and emits at a secondwavelength, while the nucleotide to be added is labeled with afluorophore that is excited at the second wavelength, but emits at athird wavelength (See, e.g., U.S. Pat. No. 7,056,661, previouslyincorporated herein). As a result, when the nucleotide and polymeraseare sufficiently proximal to each other to permit energy transfer fromthe donor fluorophore to the label on the nucleotide, a distinctivesignal is produced. Again, in these cases, the various types ofnucleotides are provided with distinctive fluorescent labels that permittheir identification by the spectral or other fluorescent signature oftheir labels.

As will be appreciated, a wide variety of analytical operations may beperformed using the overall reaction framework described herein, and asa result, are applicable to the present invention. Such reactionsinclude reactive assays, e.g., examining the combination of reactants tomonitor the rate of production of a product or consumption of a reagent,such as enzyme reactions, catalyst reactions, etc. Likewise, associativeor binding reactions may be monitored, where one is looking for specificassociation between two or more reactants, such as nucleic acidhybridization assays, antibody/antigen assays, coupling or cleavageassays, and the like.

II. Analytical Device

The analytical system in accordance with the present invention employsone or more analytical devices referred to as “optode” elements. In anexemplary embodiment, the system includes an array of analytical devicesformed as a single integrated device. An exemplar of a suitable optodeelement is disclosed by U.S. Provisional Application No. 61/306,235filed on Feb. 19, 2010, and entitled Integrated Analytical Devices andSystems (the '235 application), the entire contents of which areincorporated herein for all purposes by this reference. The exemplaryarray is configured for single use as a consumable. In variousembodiments, the optode element includes other components including, butnot limited to local fluidics, electrical connections, a power source,illumination elements, a detector, logic, and a processing circuit. Eachanalytical device or array is configured for performing an analyticaloperation as described above.

While the components of each device and the configuration of the devicesin the system may vary, each analytical device typically comprises thegeneral structure shown as a block diagram in FIG. 1. As shown, ananalytical device 100 typically includes a reaction cell 102, in whichthe reactants are disposed and from which the optical signals emanate.“Reaction cell” is to be understood as generally used in the analyticaland chemical arts and refers to the location where the reaction ofinterest is occurring. Thus, “reaction cell” may include a fullyself-contained reaction well, vessel, flow cell, chamber, or the like,e.g., enclosed by one or more structural barriers, walls, lids, etc., orit may comprise a particular region on a substrate and/or within a givenreaction well, vessel, flow cell or the like, e.g., without structuralconfinement or containment between adjacent reaction cells. The reactioncell may include structural elements to enhance the reaction or itsanalysis, such as optical confinement structures, nanowells, posts,surface treatments, such as hydrophobic or hydrophilic regions, bindingregions, or the like.

In various respects, “analytical device” refers to a reaction cell andassociated components that are functionally connected. In variousrespects, “analytical system” refers to one more associated analyticaldevices and associated components. In various respects, “analyticalsystem” refers to the larger system including the analytical system andother instruments for performing an analysis operation.

In some cases, one or more reactants for the reaction of interest may beimmobilized, entrained or otherwise localized within a given reactioncell. A wide variety of techniques are available for localization and/orimmobilization of reactants, including surface immobilization throughcovalent or non-covalent attachment, bead or particle basedimmobilization, followed by localization of the bead or particle,entrainment in a matrix at a given location, and the like. Reactioncells may include ensembles of molecules, such as solutions, or patchesof molecules, or it may include individual molecular reaction complexes,e.g., one molecule of each molecule involved in the reaction of interestas a complex. Similarly, the overall devices and systems of theinvention may include individual reaction cells or may comprisecollections, arrays or other groupings of reaction cells in anintegrated structure, e.g., a multiwall or multi-cell plate, chip,substrate or system. Some examples of such arrayed reaction cellsinclude nucleic acid array chips, e.g., GeneChip® arrays (Affymetrix,Inc.), zero mode waveguide arrays (as described elsewhere herein),microwell and nanowell plates, multichannel microfluidic devices, e.g.,LabChip® devices (Caliper Life Sciences, Inc.), and any of a variety ofother reaction cells. In various respects, the “reaction cell”,sequencing layer, and zero mode waveguides are similar to thosedescribed in U.S. Pat. No. 7,486,865 to Foquet et al., the entirecontents of which are incorporated herein for all purposes by thisreference.

Although the exemplary analytical device includes an array of analyticaldevices having a single waveguide layer and reaction cell layer, onewill appreciate that a wide variety of layer compositions may beemployed in the waveguide array substrate and cladding/reaction celllayer and still achieve the goals of the invention (see, e.g., publishedU.S. Patent Application No. 2008-0128627, incorporated herein for allpurposes by this reference).

The analysis system typically includes one or more analytical devices100 having a detector element 120, which is disposed in opticalcommunication with the reaction cell 102. Optical communication betweenthe reaction cell 102 and the detector element 120 may be provided by anoptical train 104 comprised of one or more optical elements generallydesignated 106, 108, 110 and 112 for efficiently directing the signalfrom the reaction cell 102 to the detector 120. These optical elementsmay generally comprise any number of elements, such as lenses, filters,gratings, mirrors, prisms, refractive material, or the like, or variouscombinations of these, depending upon the specifics of the application.

In various embodiments, the reaction cell 102 and detector 120 areprovided along with one or more optical elements in an integrated devicestructure. By integrating these elements into a single devicearchitecture, one improves the efficiency of the optical couplingbetween the reaction cell and the detector. In particular, inconventional optical analysis systems, discrete reaction vessels aretypically placed into optical instruments that utilize free-space opticsto convey the optical signals to and from the reaction vessel and to thedetector. These free space optics tend to include higher mass and volumecomponents, and have free space interfaces that contribute to a numberof weaknesses for such systems. For example, such systems have apropensity for greater losses given the introduction of unwanted leakagepaths from these higher mass components, and typically introduce higherlevels of auto-fluorescence, all of which reduce the signal to noiseratio (SNR) of the system and reduce its overall sensitivity, which, inturn can impact the speed and throughput of the system. Additionally, inmultiplexed applications, signals from multiple reaction regions (i.e.,multiple reaction cells, or multiple reaction locations withinindividual cells), are typically passed through a common optical train,or common portions of an optical train, using the full volume of theoptical elements in that train to be imaged onto the detector plane. Asa result, the presence of optical aberrations in these opticalcomponents, such as diffraction, scattering, astigmatism, and coma,degrade the signal in both amplitude and across the field of view,resulting in greater noise contributions and cross talk among detectedsignals.

The devices of the invention, in contrast, include relatively lowvolumes between the reaction cell and the detector, thereby reducing thenoise contributions from those components, and provide few or no freespace regions between optical components that can contribute to lossesand necessitate the use of small numerical aperture detection. Further,in preferred aspects, a given reaction region is provided with its owndevoted optical train to direct signals to a devoted portion of thesensor.

In various embodiments, the device is configured such that emitted lightfrom the fluorescent species in the nanoscale well is transmittedthrough a solid medium (e.g., a substantially solid medium), and nottransmitted through only free space (e.g., an air gap) on its way to thedetector. A substantially solid medium includes a medium with regions ofboth solid medium and air. In an exemplary embodiment, the substantiallysolid medium is a multilayered dielectric including one or more solidlayers and, optionally, one or more air layers. The substantially solidmedium is generally transparent to the emitted fluorescent light. Thesolid medium can comprise inorganic or organic materials, comprising,for example a metal oxide, glass, silicon dioxide, or transpiringpolymeric materials. While generally transmitting the emittedfluorescent light, the optical layer between the nanoscale well and thedetector can also be configured to act as a filter to other portions ofthe electromagnetic spectrum. For example, the optical layer cancomprise one or more filter layers that block or reflect unwantedportions of the spectrum. Such filters can comprise dichroic filters ordielectric stacks comprising layers of materials having differentrefractive indices. In some cases, these dichroic filters can have thinlayers comprising air in order, for example to provide a low refractiveindex layer. While the optical layer may comprise a thin layercomprising air, it is to be understood that the material having one ormore of such regions is still a substantially solid medium, and thatsuch a thin layer or series of layers would not constitute the use offree space optics. The thin layer comprising air has a thickness that isgenerally greater than about 0.1 micron, 0.2 micron, 0.5 micron or 1micron. The thin layer comprising air has a thickness that is generallyless than about 100 micron, 50 micron, 20 micron or 10 micron. The thinlayer comprising air has a thickness that is generally from about 0.1micron to about 100 micron, between 0.5 micron and 50 micron, or betweenabout 1 micron and 10 micron.

As a result, optical aberrations are confined to individual reactionregions, as opposed to being applied across an entire array of reactionregions. Likewise, in a further preferred aspect, the reaction region,optical train, and detector, are fabricated in an integrated process,e.g., micromechanical lithographic fabrication processes, so that thecomponents are, by virtue of the manufacturing process, pre-aligned andlocked in to such alignment by virtue of the fabrication process. Suchalignment is increasingly difficult using free space optics systems asreaction region sizes decrease and multiplex increases. In addition, byintegrating such components into one unified component, relativemovement between such sub-components, as is the case with free spaceoptics, can make drift and continued alignment resulting fromvibrations, a more difficult task. Likewise, the potential forcontamination in any of the intermediate spaces (e.g., dust and/or othercontaminants) is eliminated or at least substantially reduced in anintegrated system, as compared to free space systems.

In addition to reducing noise contributions from the optical pathway,the integrated devices of the invention also benefit from fabricationprocesses and technology that eliminate other issues associated withdiscrete reaction cell, optic, and detection components. For example,with respect to certain highly multiplexed or arrayed reaction cells,initial alignment and maintaining alignment of the detection with thereaction cell over the full length of the analysis can raisedifficulties. This is particularly the case where excitationillumination may be specifically targeted among different arraylocations of the reaction cell and/or among different reaction cells.

In the embodiment shown in FIG. 1, a signal source, a transmission layercomprising optical components to modulate the light transmittedtherethrough, and a detector are joined together into an integrateddevice.

As used herein, the term “integrated” may have different meanings whenused to refer to different aspects of the invention. For example, in thecase of an integrated optical device or an integrated optical system,the term integrated generally means that the various components arephysically connected, and that the optical signals pass from componentto component through solid media. The optical signals generally travelwithout passing into significant regions of air or free space, as wouldbe understood by one in the field of optics. The integrated opticalsystem may have regions comprising thin films comprising air, forexample in the context of a dielectric stack or dielectric filter asdescribed herein. In the context of the description of a system, theterm “integrated” is to be understood as generally used in theanalytical and electrical engineering fields, where “integrated” wouldrefer, for example, to a combination or coordination of otherwisedifferent elements to provide a harmonious and interrelated whole,whether physically or functionally. The meaning of the term willgenerally be understood by one of skill in the art by the context inwhich it is used.

Being an integrated device, the light emitted from the reactor cell 102will pass through to the detector through a solid medium. In someembodiments, the integrated analytical device also comprises componentsfor providing illumination to the reactor cell 102. For example, in manycases where reactor cell 102 comprises a zero mode waveguide, it isoften desirable to provide illumination from below the reactor cell, forexample between the bottom of reactor cell 102 and the transmissionlayer or optical train 104. In some cases, a waveguide structure isincorporated into the analytical device to provide such illumination.Analytical devices comprising waveguides for illumination are describedin more detail herein, and for example, in U.S. patent application Ser.No. 11/849,157 filed Aug. 31, 2007 and Ser. No. 12/560,308 filed Sep.15, 2009, which are incorporated herein by reference for all purposes.

In various embodiments, the analytical device is a substrate including areaction cell array, and a detector array on a bottom surface of thearray. The device may also include other components such as processingcircuits, optical guides, and processing circuits. In variousembodiments, the analytical device may be formed by building layers on asubstrate or by bonding two or more substrates. In an exemplary device,a fused silicon (FuSi) substrate, a ZMW layer, and a silicon substratewith a photodetector array are bonded together to form the array ofanalytical devices. One will appreciate that such integrated analyticaldevices have significant advantages in terms of alignment and lightcollection. For example, the reaction site and detector are alignedthrough the manufacturing process. One will appreciate from thedescription herein, that any of the components and systems may beintegrated or modified in various manner. In another example, the ZMWsubstrate and detector array are on separate substrates that are broughttogether for the experiment, after which the ZMW substrate is replacedwith another substrate for a second experiment. With this approach, thedetector array may be re-used rather than being disposed with the ZMWsubstrate after an experiment. It may also be more cost effective as theyields from each of the processes are separated. In this manner, the ZMWarray and detector array are in intimate contact during the experiment(as if they are part of an integrated device), but they can be separatedafter the measurement.

An example of a device that includes integrated reaction cell, sensorand optical components including an illumination conduit is shown inFIGS. 1B and 1C. As shown in FIG. 1B, the integrated device 700, shownfrom an end view, includes a plurality of reaction cells 702. Eachreaction cell 702 is in optical communication with an optical conduit orwaveguide 706, that is disposed in a waveguide array substrate layer 704which delivers excitation illumination to the reaction cell. Opticalsignals emitted from the reaction cell are then conveyed from thereaction cell 702, through the waveguide array substrate 704, to becaptured and relayed by integrated optical elements, e.g., opticaltunnels 708, to deliver the signals to the sensor elements 710 of sensorarray 712. A top down view is shown in FIG. 1C which schematicallyillustrates the separate waveguides 704, the separate reaction cells702, and the separate sensor elements 710 on the detector array 712 (notshown). Illumination is delivered through one end of the waveguides 704and is propagated along the length. Because the reaction cells aredefined in a substrate layer overlaid onto the waveguide array substrate706, that substrate layer operates as a cladding layer everywhere butwhere the reaction cells are defined. For example, in the case of ametal clad zero mode waveguide array, the reaction cells are defined ina metal layer that forms an upper cladding to the waveguides in thewaveguide array substrate 706. At the location where the reaction cellsare defined, the cladding is not present, allowing evanescentillumination of the reaction cell from the underlying waveguide 704. Aswill be appreciated, a wide variety of layer compositions may beemployed in the waveguide array substrate and cladding/reaction celllayer, and still achieve the goals of the invention (see, e.g.,published U.S. Patent Application No. 2008-0128627, previouslyincorporated herein). In some cases, the cladding for the zero modewaveguides is not a metal, but is a material having a refractive indexthat is lower than the index of refraction of the transparent layerbelow it. The refractive index of the cladding is generally chosen suchthat there will be total internal reflectance of the incomingillumination. The refractive index difference between the cladding andthe transparent layer below the cladding will depend on factors such asthe wavelength of light that is used for illumination and the angle atwhich the illumination light strikes the surface between the claddingand the transparent layer. Where the angle of incidence of theillumination light is shallow, the refractive index difference requiredfor total internal reflection can be smaller. Selection of theappropriate refractive index difference for total internal reflection iswell known in the art. Where a material having a lower refractive indexis used as a cladding, it can be useful to have the refractive index ofa fluid within the zero mode waveguide be close to the refractive indexof the cladding in order to minimize any scattering from the zero modewaveguide structure. In some cases, the refractive index of the fluid issubstantially the same as the refractive index of the cladding. In somecases the difference in refractive index is less than 0.001, or lessthan 0.0001. In some cases the difference in refractive index is between0.01 and 0.00001.

The size of the processing circuits in each of the analytical devicesmay be minimized to reduce costs. By developing a board in the receivercamera electronics (e.g. massively parallel DSP or microprocessor or adedicated FPGA, CPLD or ASIC), overall operating costs (i.e.$/mega-base) may be minimized.

Another embodiment of an integrated analytical device of the invention(optode) is shown in FIG. 2A. While FIG. 2A is shown in open form toillustrate the various components, it is to be understood thatanalytical device of FIG. 2A represents a structure that comprises allsolid or liquid components, and that there is no substantial open orfree space between the components.

An additional illustration of a device and system integration asdescribed herein is shown in FIGS. 2A and 2B. As shown in FIG. 2A, theanalytical device has a reaction cell 802 that is coupled with a reagentreservoir or fluid conduit 806 which delivers reactants to the reactioncell 802. The reaction cell can be a nanoscale well or zero modewaveguide. In some cases, the reaction cell will have a biomolecule suchas a polymerase enzyme immobilized within it. The fluidic conduit canprovide reagents across a number of reaction cells. Below the reactioncell is a waveguide for providing excitation illumination to thereagents within the reaction cell. While a waveguide is shown here,other optical elements such as those provided elsewhere herein can beused to provide light from under the reaction cell. The illuminationlight can be used to excite fluorescent emission from reagents with thereactor cell. The light emitted from the reaction cell is directeddownward through a transmission layer, which acts to transmit the lightfrom the reaction cell to the detector. In some cases, the transmissionlayer will have optical components to enhance the efficiency of thelight transfer or modulate the light. In the analytical device of FIG.2A, an optical tunnel or conduit 808 is disposed in opticalcommunication with the reaction cell 802, which is in turn in opticalcommunication with sensing element(s) 810 in the detector, shown as amulticolor discriminating set of sensor elements. The sensor elementsare coupled to appropriate electronic components, such as busses andinterconnects 812, that make up the overall sensor or camera. Also shownis a waveguide 814 for delivery of excitation illumination to reactioncell 802. FIG. 8B shows a larger view of a multiplexed device includingmultiple reaction cells and associated components, such as arrayedwaveguides 814. Also shown are fluidic conduits 816 also integrated intothe device and disposed in fluid communication with the various reactioncells. The overall device 800 is shown schematically coupled to aprocessor 820 and a computer 822. In some cases, the detector hasmultiple sensing elements, each for detecting light having a differentcolor spectrum. For example, in the case of sequencing, the sensor foreach reaction cell can have 4 elements, one for each of the four bases.In some cases the sensor elements provide color discrimination, in othercases, color filters are used to direct the appropriate color of lightto the appropriate sensor element shown as a multicolor discriminatingset of sensor elements in FIG. 2A. The sensor elements are coupled toappropriate electronic components 812, such as busses and interconnects,that make up the overall sensor or camera. The electronic components canalso include processing elements for processing the signal from thedetectors.

III. Optode Arrays and Packaging

The integrated analytical devices of the invention are generallyfabricated into arrays of devices, allowing for simultaneously observingthousands to millions of analytical reactions at one time. These arraysof optodes generally require the input of fluids to provide reagents andthe conditions necessary for carrying out analytical reactions, theinput of excitation light for the measurement of fluorescence, andconnections for the output of signal data from the detectors. Theinvention provides devices, systems, and methods for packaging theoptode arrays for these inputs and outputs.

FIG. 3 provides an embodiment for providing an array of optode elementsinto an optode array group and an optode array chip, which facilitatethe input of light and fluid and the electronic output of data. Theoptode array chip can be introduced into an instrument or system that isconfigured with the required input and output connections. For example,in some cases the light and fluid can be introduced from above throughinput ports on the top of the chip, and electronic data can be extractedfrom below the chip from electronic contacts on the bottom of the chip.The optode array group comprises an optode array component 310, afluidic input component 320, and an illumination input component 330. Inthe embodiment shown in FIG. 3, the fluidic input component 320 andillumination input component 330 are attached to the optode arraycomponent at the edges.

The exemplary optode array component 310 comprises an array of optodeelements. The number of optodes in the array can be set by thecharacteristics of the analytical reaction to be measured. The number ofoptode elements in an optode array component can be from about 10 toabout a million or more. In some cases the number of optode elements isfrom about 100 to about 100,000. As shown in FIG. 3, the fluidic conduitextends over a given optode to the optodes on other sides. As shown inthe figure, the exemplary fluidic conduit extends across the optodeelement in one direction, but essentially not in the perpendiculardirection. The fluidic conduits can be fashioned in some cases to extendover multiple optode elements in either or both directions. In somecases, the conduit can deliver fluid to all of the optodes on the optodearray component. In some cases, one conduit can deliver fluid to asubset of the optode elements, while other conduits deliver fluid toother optode elements. In some cases, each conduit delivers fluid to asingle optode element. Analogously, the waveguides shown for a singleoptode element in the figure generally extend across multiple optodeelements in the array. The waveguides can be channel waveguidesextending down a single row of optode elements providing illumination tothe reaction cells in that row, or the waveguides can be channelwaveguides wider than one row, illuminating more than one row. Thewaveguides can also be planar waveguides illuminating sections orilluminating all of the reaction cells in the optode array component.

The fluidic input component 320 has a fluid input port 322 forintroduction of fluids to the optode array chip. In the embodiment shownin FIG. 3, the fluid input port is accessible from the top. The fluidicinput port 322 has a number of fluidic conduits that extend from theinput port to the optode array component. The fluidic conduits on thefluidic input port generally mate directly with the fluidic conduits onthe optode array component, and both components are generally formed inthe same set of process steps. The number of fluidic conduits may dependon the application. In some cases, one fluidic conduit will providefluid for one row within the reaction cells in the optode arraycomponent.

The illumination input component 330 has an illumination input port 332such as a light pipe for the input of illumination light onto the optodearray chip. The illumination input port 332 is connected to a pluralityof waveguides that extend from the illumination input port into thewaveguides on the optode array. Briefly, waveguides may be providedwithin the substrate by including higher IR regions to convey lightthrough a lower IR material substrate, where the lower IR materialfunctions as a partial cladding for the waveguide. The waveguide meetsthe reaction cell with an absence of cladding, allowing evanescentillumination of the reaction cell from the waveguide.

The combination of an optode array component 310, a fluidic inputcomponent 320, and an illumination input component 330 as shown in FIG.3 can be referred to as an optode array group. A plurality of optodearray groups can be combined to form an optode array chip. The optodearray chip can comprise from 1 to about 100, from about 100 to about1,000, or more optode array groups. The optode array chip comprisingmultiple optode array groups can be fabricated using semiconductor andmicrofabrication processing techniques. For example, an optode arraychip containing an array of optode array groups can be fabricated on awafer, and the wafer can be diced into smaller optode array chips havingthe appropriate number of optode array groups for a particularapplication. The optode array chips thus produced will have the fluidicand illumination input ports, and will have electrical contactsextending from the detectors and other electronic elements on the chipfor the transfer of data.

FIG. 4 illustrates how an optode array component (middle) having, forexample, 200 by 200 optode elements can be supplied with fluid and lightfrom the side. Fluidic port 33 c dispenses fluid into an array offluidic channels that bring the fluid to the reaction cells or ZMWs.Light pipe 53 c couples light into channels that transfer theillumination light into the reaction cells from below. Emitted lightfrom the ZMWs is transmitted through a transparent transmission layerdown to the detector, in this case a photodiode. The photodiodes detectoptical signals and transmit data signals into image processing elementson the chip. Processed signal data is sent to computers for furtherprocessing through the electrical contacts on the bottom of the chip.The image processing elements on the chip are useful for processing thedate as it comes from the photodetectors. In general, for nucleic acidsequencing, the rate of optical events is relatively low, e.g. on theorder of 10³ per second. This rate is significantly lower than thetypical rate of processing that an the image processing elements arecapable of working at, which can be on the order of 10⁹ per second. Oneapproach that can be employed as part of the invention is to have asingle image processing element process data from multiple pixels, e.g.from 10 to 1000 pixels. This approach allows for maximum utilization ofthe image processing elements, e.g. transistors.

In one aspect, the invention comprises a device comprising an array ofoptode elements wherein each optode element has a reaction cell such asa ZMW or a nanoscale aperture within a cladding layer, the reaction cellconfigured to receive fluid that contains the reactive species to beanalyzed. The analysis generally comprises at least one fluorescentlylabeled species, the fluorescence from which will provide informationabout the reaction. Above the reaction cell is a fluidic layer that isin fluid communication with the reaction cell. Below the aperture layeris a waveguide layer that provides illumination to the nanoscale wellwith evanescent irradiation. The waveguide layer can comprise channelwaveguides and/or planar waveguides. Below the waveguide layer is atransmission layer that transmits light emitted from the fluorescentspecies in the reaction cell to the detector below. Below thetransmission layer is a detector layer which receives and detects theemitted light transmitted through the transmission layer, wherein theemitted light is transmitted to the detector without being transmittedthrough air. In some cases, the detector layer has below it electricalcontacts for transmitting data signals out of the chip into computercomponents for analysis and processing. In some cases processingelements are built into the chip to provide some processing of thesignals before sending the data off of the chip.

The array of optode elements is generally provided in one integrated,solid package. In some cases, the portion of the array of optodeelements that comprise the detector can be reversibly separated from theportion of the array comprising the reaction cell. This allows for thedetector portion to be used over and over again with different arrays ofreaction cells.

IV. Measurement Systems Comprising Optode Arrays

The optode array chips comprising optode arrays, inputs for light andfluid, and outputs for electronic transfer of data can be inserted intostructures that provide for the analysis reaction. In some cases, theoptode array chip can be sandwiched within an assembly that providesphysical alignment of the input and output features, and can provide theforce required for effective mating of the assembly components. Oneapproach to an assembly is the use of a clamshell assembly. An exemplarysystem includes an array of analytical devices integrated into a systemwith a test socket. An exemplary system architecture makes use ofautomated testing equipment and chip-scale packaging techniques. Invarious embodiments, the test socket is an automated test equipment(ATE) socket (shown in FIG. 5A). In the exemplary system, the socket isconnected to the processing system and other system components such asthe electrical system.

In some aspects the invention provides an assembly having a sandwichstructure comprising: a top piece comprising inputs for illuminationlight and fluid; an integrated analysis chip in the middle comprising:an aperture layer comprising a plurality of nanoscale apertures througha cladding layer in fluidic contact with the top of the chip, and awaveguide layer comprising a plurality of waveguides configured toprovide illumination light to the nanoscale apertures from below, thewaveguide layer having one or more illumination ports on the top surfacefor providing illumination light to the waveguides; a transmission layercomprising a transparent material for transmitting emitted light fromthe nanoscale apertures; a detector array layer below the transmissionlayer having detectors electrically connected to pins extending out thebottom of the chip; and a bottom piece having electrical contactscorresponding to the pins on the bottom of the chip; the assemblyconfigured such that upon closure, the chip is aligned with the top andbottom pieces to allow input of the illumination light and fluid fromthe top piece and extraction of electrical signals from the bottompiece.

An exemplary integrated device isolates the electrical components fromthe optical and fluid components, for example, having the optical andfluid delivery on one side and the electrical interconnects on the otherside of the device. One embodiment of a system is shown in FIGS. 5 and5A in which an optode array chip 40 comprising, for example, an array ofoptode array groups 56, is inserted into a socket comprising a top piece51 which delivers illumination through an illumination system 53 andfluidics delivery system 33 to the optode array chip, and bottom piece57 which has an array of electrical contacts which mate with theelectrical contacts on optode array chip. In some embodiments the socketcan use vacuum to pull the components of the system together to enhancefluidic, illumination, and electrical contacts.

The electrical connections are generally on the bottom surface of theintegrated device and optical and fluidic connections on the top side ofthe device (shown in FIG. 5). The partition of the electrical componentsin this exemplary manner provides for a two-sided socket that can supplyall 110 connections within standard commercial tolerances. As anexample, the clamshell socket used in the exemplary commercial ATE maybe modified to be used with the analytical array 40. Such test socketsgenerally have over 50,000 insertion cycle reliability and provideadequate and uniform contact force. Moreover, because the components areintegrated into a single device and the socket is self-seating, theoptical components and detector are automatically aligned. The exemplaryincludes spring loaded durable contact pins and oxide scrubbing crownsto further promote auto alignment and reliable contact. Thus, theintegrated device can be easily connected to the processing system andother system components by insertion into the socket. This provideshigher reliability, lower cost, and generally easier use by thetechnician.

The reagent handling, sample handling, and illumination functions may beperformed in a distributed manner on an area above a processing regionof the integrated device and adjacent to the reactor cells (shown, forexample, in FIG. 13B). The illumination ports and fluidics ports may bepositioned in alternating rows in a checkerboard pattern. Theseillumination and fluidic ports can service either a single adjacentoptode array component 56 or in some cases can service four of thenearest neighboring optode array components. The distribution ofillumination and fluids is more uniform, less complex, and performanceis maintained to very high multiplex via array segment scalability. Eacharray segment illumination and fluidics can be individually controlledif desired. In various embodiments, fluidics and photonic connections tosocket 51 are made on the top portion of device 40.

Referring to FIG. 5, a sample is provided to the top of the socket andintroduced to a set of pipettes that are aligned with the fluidic portson the optode array. Since the optodes are grouped into sub-arrays, thereduced number of fluidic ports allows for alignment to standardcommercial tolerances (e.g. about 0.3 mm) and the reduced number ofconnections increases reliability. The failure of a single port does notmake the entire experiment invalid and the remaining ports can collectdata.

The integrated system of the present invention is typically configuredto introduce fluids and optical signals. To provide for a sterileenvironment to introduce sample and reagent, a low cost fluidicdistribution device with single-use capability can be inserted into thesocket with each experiment. This fluidic device can be molded withstandard bio-compatible polymers similar to multiple micro-pipettesystems sold by companies such as Biohit, Thermo and Eppendorf. Anexample of a disposable 2-D micro-pipette insert for the ATE clamshellsocket lid is shown in FIG. 6. FIG. 7 shows a diagram of theintroduction of fluids into the optode array chip with an array ofmicropipettes configured to mate with the fluidic input ports on anoptode array chip. The micropipette array 83 mates with fluidic inputports 82 on the optode array chip. The fluid extends down conduits 86into the optode elements. The ZMWs within the optode elements areilluminated, and emitted light is transmitted through light pipes to thedetectors 84. The detectors send signals to data processing componentswithin the chip.

The introduction of fluidics to optode groups may be done withhomogenous material, or alternatively, each optode group could beoperated with a different sample or reagent setup to perform highlymultiplexed assay experiments. The temperature of each fluidic input canalso be adjusted or maintained, for example, to provide variability inthe assay.

In various embodiments, the introduction of the photonic illuminationsignal is accomplished with discrete light ports at the top part of theclamshell socket within commercial tolerances (e.g. between about 0.3 mmto about 0.6 mm). By distributing the light energy in the durable socketto local optode regions, careful design and exotic materials can be usedto minimize losses, enable polychromatic excitation and reduce heat loadon the active single use device. For example, a lithium niobatewaveguide structure can be designed with very low insertion andpropagation losses to the optode group. Lower quality distributionnetworks on the disposable chip are enabled as the transmission distanceand branching are significantly reduced. The photonic distributionnetwork can be developed to be interleaved with the microfluidicdistribution insert as shown in FIG. 8 and FIG. 9.

In various embodiments, a top-side flood illumination method is used asshown in FIG. 10. The fluidics channels and ZMWs are optically shieldedfrom topside illumination and a path to direct the light to the activearea of the ZMWs is provided. The top half of the exemplary ATE socketis transparent to the flood illumination while shielding themicrofluidic insert. The socket may be made of a waveguiding material toassist the flood illumination of the part. For example, the socket mayinclude a structure or materials selected to guide the floodillumination along a predetermined path. In FIG. 10 the optode arraychip comprises an array of ZMWs 91 formed within a transparent substratesuch as glass. Surrounding the ZMWs and extending into the top surfaceof the glass substrate are regions of opaque cladding material,comprising, for example, a metal such as aluminum. Illumination light 96introduced from above the chip passes down through the glass and isdirected upward to the bottom of the ZMWs by optical elements 95, whichcan comprise mirrors or dielectric stacks. The light can stimulateemission from sample within the ZMW, the emitted light from which istransmitted down to optical detectors 97. Fluid is transported to theZMWs by fluid conduits 92. The tops of the ZMWs are covered with anoptically opaque covering 94 which prevents the illumination light fromentering the ZMW from above. The opaque covering 94 can comprise a metalsuch as aluminum. In some embodiments, top illumination is carried outwith a structure similar to that shown in FIG. 10 wherein each ZMW 91 isilluminated from above, and the optical element 95 includes a reflectivestructure that is circularly symmetric with respect to the ZMW. Acatoptric type arrangement can be used for reflecting the light thatenters from above the chip up into the ZMW after reflecting off of acircularly symmetric reflecting element below the ZMW. The catoptricsystem can have curved surfaces, e.g. parabolic surfaces designed tooptimize the amount of light impinging on the ZMW. The center of thecatoptric would be non-reflective, allowing light emitted from the ZMWto pass through to one or more photodetectors disposed below thereflective element. The relative size of the opening is chosen toprovide the best balance between high intensity illumination, andmaximum amount of light collection.

In some aspects, the invention comprises a device for measuringanalytical reactions comprising a transparent substrate comprising aplurality of rows of nanoscale apertures extending through an opaquecladding to the top of the transparent substrate. The rows of nanoscaleapertures are separated by regions of the transparent substrate open toillumination from above. The device has a plurality of fluidic conduits,each on top of and in fluidic contact with a row of nanoscale apertures.For these exemplary devices each fluidic conduit is coated with anopaque material that prevents the illumination light from entering thenanoscale aperture from above. In addition, the device has a series offeatures below the nanoscale apertures configured to direct illuminationlight from above the transparent substrate up into the nanoscaleapertures from below. In some embodiments the device also has built-inoptical detectors, with at least one detector per nanoscale aperture. Insome cases, the device has multiple detectors for each nanoscale well,for example, four detectors, each sensitive to a different color toallow for four color nucleic acid sequencing.

In an exemplary system, the development of low cost packaging foranalytical arrays is enabled with the use of chip scale packagingtechniques. For example, the use of through-hole vias with distributedprocessing and data collation circuitry enables the multiplexing of manyanalytical signals onto a greatly reduced number of I/O lines. Byexample, a collection of 256×256 elements each operating at 25incorporations per second and providing 5 bytes per event requires anelectrical bandwidth of about 65 mega-bits per second. This bandwidthcan be provided at only about 10% of the maximum data rate of standardLVDS signaling (ANSI-644) which only needs two connections. For a devicecapable of mapping an entire genome in 15 minutes, for example, as fewas 14 LVDS electrical connections are required as is shown in FIG. 11.

In some embodiments, a plurality of devices are formed in a substrate(e.g. wafer) cut from a sheet material. The wafer can comprise, forexample, silicon or fused silica. The exemplary device includes areal-time sensing structure integrated with the chemical reaction cellsand provides for the decoupling of the reactor location with the opticalelements. The detector elements are grouped around distributedprocessing cells thereby enabling significant performance advantageswith high parallelism. In addition, this architecture reduces thedistribution path for fluidics, signal, and stimulus by arranging cellsinto groups of manageable I/O “pads” corresponding to optode groups.

The implementation of integrated sensing elements with the cellarrays/reactors provides many benefits including higher speed operationand the ability to extract tagged signals from reduced emissions withsynchronized light. FIG. 12 shows another embodiment of an integratedanalytical device cell with a fully contained light source, cell reactorelement, and detector. By eliminating common and redundant illuminationand detection paths, the fidelity of the sensed signal is maintained.

While there are many benefits of a distributed architecture, thedistribution branching network required for a high resolution arraypresents some challenges and limitations. For example, the lossesassociated with a waveguide operating with many branches and taps willintroduce a light intensity gradient across the device. One method ofovercoming this problem is with cross-hatched, alternating waveguides.In some cases, the device uses monochromatic illumination and detectiontechniques to avoid or mitigate such problems.

Turning to FIGS. 13A, 13B, and 13C, an array with distributed functionsis shown. FIGS. 13A, 13B, and 13C represent layers within an optodearray chip illustrating the various functions performed in differentportions of the chip. FIG. 13B shows the topside of an optode array chiphaving reactor array components 71, illumination input components 72,and fluidic input components 73. FIG. 13A shows the layer in which theoptode array components have an array of detectors 70. As illustrated,the detectors are connected to processing components 75. Theseprocessing components process the signal from the detectors beforesending the signal on for further processing and analysis. FIG. 13Cshows the base of the optode array chip. The base has an array ofelectrical connections. In the embodiment shown, the portions of thechip under the optode array components have contacts for the input ofpower. The portions of the chip under the fluidic input and illuminationinput components have electrical contacts for the output of signal fromthe signal processing elements.

The illustrated array is manufactured using techniques similar tosilicon wafer preparation and testing techniques. The array is built upfrom a substrate with any of the above mentioned analytical elements.The array does not require regular spacing. One will further appreciatethat the system architecture can be easily set up and scaled. Each“unit” may be an integrated, local system with a number of optical,detection, and processing elements. The outputs of each of the reactorcell detectors (containing the preprocessed pixel data) is connected toa processing circuit where many functions of various utilities can beperformed including, but not limited to, data reduction, digitization,buffer storage, bus arbitration, and the like.

Referring to FIG. 13B, the reagent handling and illumination can beperformed in a distributed manner using the area above the processingregion and adjacent to each of the respective reactor cells. Acheckerboard pattern of alternating rows of illumination ports andfluidics ports is provided. These fluidic and illumination ports can beprovided as described above as arrays of optode array groups. Theseports can service either the single adjacent reactor array or aplurality of arrays. In various embodiments, each node or set of portsservices the neighboring arrays (e.g. arrays on each of the four sides).In contrast to conventional devices, the distribution of illuminationand fluids is more uniform and less complex and performance ismaintained to very high multiplex via array segment scalability. Onewill appreciate that each array segment illumination and fluidics can beindividually controlled if desired.

Referring to FIG. 13C, the readout of array segments can be performedvia local through-hole vias to substrate connections. The packaging andtesting of the system can be done with industry accepted and verifiedprocesses. To complement the fluidic and illumination connections on thetopside of the wafer, a number of electrical connections representingthe I/O of the array segments may be made on the bottom of the wafer asdiscussed above. These connections can be segmented by power and signalgroups.

With this top-bottom connection set-up, a standard clamshell packagingtechnique (e.g. ATE socket) as described above can be used to connectthe device to the overall system. Referring to FIG. 4, the topsideconnections involve the alignment of multiple illumination light pipes53 c and microfluidic nozzles 33 c. For example, if a 2000×2000 cellarray is needed and 100 array segments are placed in 200×200 multiplexon 5 micrometer centers, the adjacent 100 I/O and processing segmentsare about 1 mm×1 mm in size. Therefore, 10×5 connections of bothillumination and fluidics are needed but have achievable alignment atthe pitches described. In a similar fashion, the data reductionperformed in the processing regions reduces the number of electricalconnections that need to be interfaced to the external circuitry.Standard electrical bump bonds can be used to connect with standarddurable electrical sockets with achievable tolerances for high speedoperation.

Turning back to FIG. 14, the scalability of the integrated devices isextended to a scalable array segment and very high resolution. In thishigh resolution array, the performance across the array (peripheryversus center) is made more uniform with the herein-described systemarchitecture.

One will appreciate that the size and arrangement of the reactor arraysand optodes is relatively flexible. The partition of the reactor arraysections and the adjacent distribution and processing regions can besized across a relatively wide range and each section can be spaced withrespect to each other at varying distances to support the overallfunction required. Exemplary partitions are shown in FIG. 14. In FIG.14(A) an optode array component 201 a is connected to two fluidic inputcomponents 202 a and two illumination input components 203 a. In FIG.14(B) an optode array 201 a component is connected to one fluidic inputcomponent 202 and one illumination input component 203 a. In FIG. 14(C)an optode array component 201 a is connected along one edge to a fluidicinput component 202 a and along another edge to an illumination inputcomponent 203 a.

Although in various respects the analytical device is described as beingfabricated in a monolithic fashion, such that all integrated elementsare fabricated from the outset into the same structure, one willappreciate from the description herein that other manufacturingtechniques may be utilized. In some cases, different components arefabricated separately, followed by integration of the separate partsinto a single integrated device structure. For example, the sensorelements, optionally including one or more optical elements, may befabricated in a discrete component part. Likewise, the reaction cellsmay be fabricated in a discrete component part optionally along with oneor more optical components. These two separate parts can then be matedtogether and coupled into a single integrated device structure where thesensor elements in the first component part are appropriately alignedwith the reaction cells in the second component part. In variousembodiments, the analytical device employs modular assembly techniques.In this manner, various components can be joined, separated, andreassembled as needed. For example, the reaction cell array andwaveguide and sensor may be assembled during an experiment and thenseparated so the cell array and waveguide can be replaced for set-up ofthe next experiment.

Joining of two discrete parts may be accomplished by any of a variety ofknown methods for coupling different components in the semiconductorindustry. For example, two planar components may be joined using, e.g.,joining through Van Der Waals forces, ultrasonic welding, thermalannealing, electrostatic, vacuum, or use of other joining mechanisms,e.g., epoxide bonding, adhesive bonding, or the like. Appropriatejoining techniques include, but are not limited to, mechanical,chemical, and ionic techniques.

As discussed above, in joining separate parts it may be desirable tojoin such parts that respective functional components align between theparts. For example, where an overall device is intended to have adedicated sensor element for each reaction cell, it may be necessary toalign a part that includes the sensor elements with a part that includesthe reaction cells such that they are aligned in optical communication.Alignment may be accomplished through the use of structural alignmentelements fabricated onto the component parts as fiducials duringfabrication, e.g., pins and holes on opposing surfaces, ridges andgrooves, etc. Alternatively, in the fabrication process, differentactive regions may be provided upon the component parts such thatattractive forces are exhibited between regions where alignment isdesired.

For example, one could pattern complementary charged regions uponopposing component surfaces to result in an attractive force for thecorrect alignment. Likewise, patterning of hydrophobic and hydrophilicregions on opposing substrate surfaces, along with an aqueous joiningprocess, would yield an automatic alignment process, followed by anappropriate process step to remove any remaining moisture from betweenthe two parts. This process is schematically illustrated in FIG. 13D. Asshown, two substrates 902 and 904 are provided with hydrophobic regions906 patterned onto their respective surfaces. As will be appreciated,relatively more hydrophilic regions, e.g., corresponding tonon-hydrophobic regions 910, could also be patterned onto thesubstrates, as could a variety of other surface treatments. Theseregions are patterned so that alignment of the regions on opposingsubstrates would yield alignment of components within such substrates(step i). An aqueous layer (shown as droplets 908) is deposited upon thesurface of one or both of the substrates, which is generally repelled bythe hydrophobic regions (step ii). When the substrates are matedtogether, the aqueous layer aligns to the corresponding non-hydrophobicregions 910 on the opposing substrate (step iii). Following correctalignment, the aqueous layer is removed, e.g., through conventionaldrying mechanisms (step iv). The resulting substrates are then joinedthrough the Van der Waals forces between the chemically like (i.e.,hydrophobic) regions on their respective surfaces.

In accordance with this process, one could also readily introduce spacerelements in joining two device components, as shown in FIG. 13E. Inparticular, by providing either hydrophilic or hydrophobic spacingcomponents, e.g., nanoscale spacer elements, can be provided into thebonding steps (e.g., shown in FIG. 13D). As shown, a hydrophilic spaceris provided within the aqueous film between the opposing substrates. Thespacers localize to the hydrophilic regions and couple these regionstogether, leaving an air gap or space 914 in the bonded product. Again,as will be appreciated, hydrophobic spacer elements are also optionallyor alternatively used to align with and form bonding elements throughthe hydrophobic regions. The spacers may optionally comprise opticalcomponents, such as lenses, index matching materials, filter components,or the like, which are incorporated into the overall device and alignedin a self assembled manner during the bonding process.

V. Optical Components

In accordance with the present invention, in addition to integration ofthe sensor and reaction cell elements within a single analytical device,one or more optical components may be included within the device.Examples of integrated optical elements include, but are not limited to,directional optical elements, i.e., optical elements that alter thedirection of optical signals to direct those signals at or to a sensorelement or another optical element. Such elements include, e.g.,mirrors, prisms, gratings, lenses, and the like. By way of example, incertain cases, parabolic reflector elements or micro-mirrors areintegrated into the device to more efficiently direct optical signals ina given direction (See, e.g., U.S. patent application Ser. No.12/567,526, filed Sep. 25, 2009, incorporated herein by reference in itsentirety for all purposes). Other optical elements include spectralelements, e.g., elements that alter the spectral characteristics of theoptical signals including directing spectral components of a signal orset of signals in differing directions, separating a signal intodifferent spectral components, or the like. These elements include, forexample, dichroics, filters, gratings or prisms that separate a givensignal into spectral constituents.

In various embodiments, such optical components include containedoptical enclosures that efficiently collect photon signals emanatingfrom the reaction region and that are incident over a wide emissionangular distribution, and direction of those signals to an assignedsensor element or elements. Such self-contained enclosures typicallyprovide trapping within the chamber of substantial amounts of thephotons emitted from the reaction region, elimination of cross talkbetween reaction cells or regions that would otherwise result fromscattered signal entering adjacent sensor elements, reduction in leakagecurrent since the sensing elements can be made extremely small, reducingscattering paths and scattering elements within each optical chamber,and reducing auto-fluorescence due to the substantially reduced opticalpath mass and eliminated free-space regions.

FIG. 15 illustrates the general nature of optical signals from areaction cell in various aspects of the present invention. As shown, thereaction cell or region comprises a very low volume reaction region suchas a zero mode waveguides (ZMWs), e.g., ZMWs 202, disposed upon asubstrate 204. As shown in the exploded view, a ZMW comprises anunfilled core 206 or aperture disposed through a cladding layer 210 thattypically comprises a metal film layer. As described in, e.g., U.S. Pat.Nos. 6,917,726 and 7,486,865, the entire contents of which areincorporated herein for all purposes, the exemplary zero mode waveguidestructure is of sufficiently small dimensions that light that is atgreater than a cut-off frequency that enters into the waveguide core 208is not propagated through the core but exhibits evanescent decay throughthe core. This allows for efficient illumination of just the volume ofthe ZMW at the opening (schematically illustrated by the dashed linewithin core 206), and collection of any optical emissions that occurwithin the same volume. The result is to permit excitation of andcollection of fluorescent emission from individual molecules disposed atthe opening of the core, e.g., on a transparent base layer. Lightsignals from the reaction cell, or ZMW 202 as shown, are emitted in aLambertian distribution, as shown by arrows 212. Efficient capture ofsignals exhibiting this profile may necessitate either directionaloptics to re-direct the signals toward a detector, or provision of adetector that matches the hemispherical surface of this signal profile.

In accordance with certain embodiments of the invention, an opticalchamber is provided within the device, and particularly within asubstrate, to efficiently trap and direct optical signals to theintegrated sensor element. This aspect is schematically illustrated inFIG. 16. As shown, a reaction cell or region, such as a ZMW 302, isprovided, disposed above a substrate layer 304. A detector 306 isdisposed on or adjacent to the opposite surface of the substrate layer,which typically includes multiple sensor elements, e.g., sensor elements308 and 310. An optical tunnel 312 or conduit is provided in thesubstrate to more efficiently convey optical signals from the reactioncell 302 to the sensor element(s) 308 and 310. The optical tunnel istypically comprised of reflective material, such as an integrated metalwall layer 314, that contains the optical signals within the tunnel, orit is comprised of a material having a sufficiently different index ofrefraction that maintains the optical signals within the tunnel by totalinternal reflection. As shown, other components, such as electricalinterconnects and busses 316 and 318, for the sensor may also beprovided either within the detector layer 306 or the oxide or otherinsulator layer above it.

Fabrication of these devices with an integrated optical tunnel may becarried out by a variety of fabrication processes that are typicallyused in the semiconductor manufacture process. For example, one mayemploy a number of processes to fabricate reflective metal tunnelswithin the intermediate layer between a reaction cell and a sensorelement.

In one exemplary process, the optical tunnel portion is fabricated ontop of the detector and sensor elements or portions thereof. Forreference and ease of discussion, FIG. 17 schematically illustrates thetypical structure of two pixel elements of a CMOS sensor based ondetection by color differentiation. As shown, the overall structure 400includes the silicon photodiode elements 402 and 404 that correspond toeach pixel for the overall sensor or camera. Multiple additional layersare provided over the sensor elements, including an insulating oxidelayer 406, nitride layer 408, optional color filter layers 410 thatincludes different spectral filters 410 a and 410 b to allocatespectrally different signals to different pixels, oxide layer 412, andmicrolens layer 414. The foregoing discussion is provided for ease ofdiscussion of portions of the invention. The structure of CMOS sensorsused in the invention, or even the type of sensors employed, include,but are not limited to, CMOS sensors, CCDs, etc. Although FIG. 17illustrates a detector structure based on detection by colordifferentiation, one will appreciate from the description herein thatother detection techniques may be employed.

FIGS. 18 and 19 schematically illustrate one exemplary structure andfabrication process for an optical tunnel. As shown in FIG. 18, themetal tunnel 502 comprises a series of metal layers where each layerprovides an annular ring or border 504 and 506 having an increasingcross section so that, collectively, such layers define a convergentmetal tunnel that directs light to the sensor element 508 from reactioncell 510. FIG. 19 provides a schematic process flow for the fabricationof the structure shown in FIG. 18. As shown in step (i), an exemplarysensor array is provided, for which only a single photosensor pixel 508is shown disposed upon a substrate layer 512, with an insulating oxidelayer 514 disposed over it. A resist layer 516 is patterned over theinsulator layer 514 in step (ii) to permit partial etching throughinsulator layer 514 shown at step (iii), e.g., in a time or depthcontrolled etch process. A second resist layer 518 is patterned over theetched surface to provide a mask for the central portion of the opticaltunnel in step (iv). A conformal metal deposition step, e.g.,evaporation, then provides a first metal ring or border 504 for theoptical tunnel in step (v). An oxide layer 522 is then grown ordeposited over the structure in step (vi). In step (vii), the processesare repeated to deposit subsequent metal ring layer 506 and oxide layer524. As will be appreciated, this process may be further repeated toprovide additional layers to the metal tunnel 502. One will appreciatethat similar steps and processes can be used to manufacture any of thedevices and components described herein.

Similar fabrication processes may be employed to provide higher index ofrefraction (IR) material tunnels from the reaction cell to the sensorelement, or devices that include a hybrid of a high IR tunnel componentand a reflective (e.g. metal) optical tunnel. FIG. 20 provides aschematic illustration of a device having a higher IR material plugprovided in the intermediate substrate layer between the detector andthe reaction cell. As shown, the overall structure 600 includes adetector substrate 602, having a sensor element, such as siliconphotosensor 604, disposed thereon. Oxide insulating layer 606 isdisposed over the detector substrate. Layers 608 and 610 are providedwith regions of higher index of refraction 612. These regions are ofsufficiently high IR relative to the surrounding substrate material sothat they funnel light to the detector by virtue of maintaining totalinternal reflection within the higher IR region. By way of example, ifthe high IR region possesses an IR of e.g., 2.04, such as is the casefor a silicon nitride plug, that is disposed through and interfaced withan intermediate layer having an IR of 1.64, e.g., as in silicon dioxide,it would result in total internal reflection of any light impinging thatinterface at less than 30 degrees. As will be apparent from thedescription herein, a variety of methods are available for providinghigh IR regions precisely located within the substrate layer 608 and 610including, but not limited to, etching followed by nitride deposition,e.g., liquid phase chemical vapor deposition (LPCVD).

Other index shifting materials may be included in the fabrication of thedevice, including, for example, doped silica materials, e.g.,nanocrystal doped components or materials (See, e.g., U.S. PatentApplication No. 2007-0034833, the full disclosure of which isincorporated herein by reference in its entirety for all purposes),and/or air or other gas-filled gaps or spaces to provide index mismatchto guide optical signals.

As shown in FIG. 20, an optional additional metal wall component 616,e.g., as described with reference to FIG. 18 above, is provided closerto the reaction cell 618. This permits the direction of optical signalsfrom the reaction cell into the high IR regions at angles that are lessthan the critical angle for the interface of the high IR region and thesurrounding substrate, e.g., less than 30 degrees for the exemplarysilicon nitride/silicon oxide interface, and reduces the possibility ofcross talk among adjacent portions of the device (as schematically shownby the dashed arrows).

As will be appreciated, because the devices of the invention aregenerally amenable to fabrication using standard monolithicsemiconductor fabrication techniques, fabrication of the devices canincorporate much of the functional components that are employed for thedetector, e.g., the electrical interconnects and busses used for a CMOSsensor array, as well as the optical components, (optical tunnels,lenses, mirrors, etc.), and even the reaction cells themselves, e.g.,metal clad ZMWs. In addition, other functional elements may beintegrated using the same or similar processes, including, for example,microfluidic elements that may be integrated into the overall devicestructure, and illumination components, e.g., for delivery of excitationillumination to the reaction cells.

Also as noted previously, although generally illustrated in terms ofindividual or a few reaction cells and associated integrated opticalcomponents and sensors, it will be appreciated that the illustrationsand descriptions provided herein apply to much larger arrays of suchreaction cells. In particular, such devices may generally haveintegrated into a single device more than about 1000 discrete reactioncells, and associated optics and sensors. In various embodiments, theintegrated device includes a number of reaction cells in a rangeselected from between about 1000 and about 1 million, between about 2000and about 1 million, between about 1000 and about 100,000, between about100,000 and about 1 million, between about 1 million and about 10million, and more than 10 million. It may be desirable to select thenumber of reaction cells based on the desired application. For example,the device may include between about 1000 and about 100,000 cells forclinical testing, between about 100,000 and about 1,000,000 for adiagnostic laboratory, or more than about 1,000,000 for high throughputresearch.

In accordance with the invention, each reaction cell may have anindividual sensor element or pixel associated with it, or it may havemultiple sensor elements or pixels associated with it (particularlywhere spectral separation, direction and separate detection arewarranted). Likewise, each reaction cell may preferably have its owndedicated integrated optical components associated with it. In somecases, integrated optical components may be shared among multiplereaction cells, e.g., to apply standard filtering, to apply illuminationto multiple cells, or the like, and will typically be in addition to oneor more dedicated optical components.

As referred to above, in some cases, illumination optics are includedwithin the integrated device structure. These optics may include actualillumination sources, e.g., LEDs, solid state laser components, or thelike, and/or they may include optical conduits for transmission ofexcitation illumination from either an internal or external light sourceto the reaction cell. Examples of particularly preferred opticalconduits include waveguides integrated into the substrate adjacent tothe reaction cell. Examples of such illumination conduits have beenpreviously described in, e.g., published U.S. Patent Application No.2008-0128627, the full disclosure of which is incorporated herein byreference in its entirety for all purposes.

In various embodiments, the illumination source is reversibly opticallycoupled to the illumination ports. By “reversibly optically coupled” itis meant that one element, which is functionally coupled to anotherelement, may be removed. In other words, the coupling is not permanent.As used herein, for example, the illumination source may be connectedand disconnected from the illumination port.

As noted previously, optical cavities within the device may be useful ina variety of ways, depending upon the nature of the application andarchitecture of the device. For example, such gaps or spaces may beemployed in the optical train to provide additional signal funneling toa detector or sensor element. Alternatively, these gaps may provide anillumination conduit for delivery of illumination radiation to areaction cell.

VI. Detector Components

As noted previously, in some applications, it may be desirable todistinguish different signal components, e.g., to identify that both areaction has occurred and to identify the participants in that reaction.By way of example, in the case of nucleic acid sequencing, one canprovide different nucleotides with different optical labeling groupsthereby allowing not only detection of a polymerization reaction butalso identifying the particular type of nucleotide that was incorporatedin that polymerization reaction. Accordingly, it would be desirable toinclude the ability to distinguish different signal components withinthe devices and/or systems of the invention.

In some optical systems, the ability to distinguish different signalcomponents is achieved through the use of, e.g., different filteredoptical trains, or the inclusion of dispersive optical elements todifferentially direct different spectral components of a signal todifferent detectors or different regions on a given detector array. Invarious embodiments, the system is configured for detection anddifferentiation based on other detection techniques. Various aspects ofthe detection devices and methods are similar to those described in U.S.Patent Publication Nos. 2007/0036511 filed Aug. 11, 2005, 2007/0036511filed Aug. 11, 2005, 2008/0080059 filed Sep. 27, 2007, 2008/0128627filed Aug. 31, 2007, 2008/0283772 filed May 9, 2008, 2008/0277595 filedSep. 14, 2007, and 2010/0065726 filed Sep. 15, 2009, and U.S. Pat. Nos.7,626,704, 7,692,783, 7,715,001, and 7,630,073, the entire content ofwhich applications and patents are incorporated herein for all purposesby this reference.

In the context of integrated devices, the available space for use indifferential direction of signal components is generally reduced.Similarly, where a single sensor element is assigned to a reaction cell,one may be unable to direct different components to different detectors.

The integrated device may include directional components and/or filtercomponents that selectively direct different spectral components of asignal to different adjacent pixels or sensors within the device. By wayof example, a given reaction cell and its associated optical train mayinclude multiple individual sensor elements associated with it, e.g.,pixels. Included within the optical train would be a directionalcomponent that would direct spectrally distinguishable signal componentsto different sensor elements or collections of sensor elements. Examplesof such components include prisms, gratings or other dispersive elementsthat can redirect and separate signal components. The use of suchcomponents in optical systems is described in, e.g., published U.S.Patent Application No. 2008-0226307, the full disclosure of which isincorporated herein by reference in its entirety for all purposes.

In addition to such directional elements, or as an alternative to suchelements, multiple sensor elements may be provided with filtering opticsthat allow only a single signal type to reach that particular sensorelement. Each sensor is differently filtered to allow it to detect aparticular signal component, to permit multicolor distinction. Inparticular, each of a plurality of sensor elements within a givenreaction cell's dedicated optical train is provided with a filter thatnarrowly passes one component of the overall signal from the reactioncell. For example, the signal associated with a given nucleotideincorporation event would be passed by a filter on a first pixelelement, but rejected by the filter on three other adjacent pixelelements. Each of the different filter layers on each sensor would beselected for the given signal components for a given application.Further, each reaction cell could have one, two, three, four, or morepixel elements dedicated to receiving the signals from that reactioncell. In some cases, 5, 10, 20, 50 or even 100 pixels or more could bedevoted to a given reaction cell.

Deposition of a variable filter layer, i.e., providing different filterson different pixels or collections of pixels, may generally beaccomplished during the fabrication process for the overall integrateddevices or the underlying sensor elements using conventional CMOSfabrication processes. Likewise, dichroic filters are equally amenableto fabrication/patterning onto the sensor elements to reject anypotential excitation illumination.

Alternatively, or in addition to selective direction/filtering of theoutput signals from a reaction cell, distinguishing signal componentsmay also be accomplished by detecting an output signal in response to aspecific excitation event. In particular, if a signal is received inresponse to an excitation radiation that is specific for a given signalgenerator, e.g., fluorescent label, one can assume that the label ispresent. By modulating or interleaving the excitation illuminationacross the excitation spectra for multiple fluorophores having differingexcitation spectra (or different excitation/emission profiles), one canidentify when any of a set of fluorophores is present in the reactioncell. By correlating an emitted signal with a given excitation event,one can identify the fluorophore emitting the signal. Examples of thisprocess are described in published U.S. Patent application No.2009-0181396, the full disclosure of which is incorporated herein byreference in its entirety for all purposes. As will be appreciated, thetiming of illumination, the frame rate of the detector, and the decaytimes for the fluorophores are matched to provide optimal detectabilityof each different signal event, without different events bleeding overinto each other, while also permitting sufficient sampling during agiven frame capture event for the detector, that no individual eventsare missed.

In an exemplary process, a given application that includes multipledifferent labeled species, e.g., different labeled nucleotides, includeslabels that differ in their excitation spectra. Illuminating a reactionmixture iteratively with the different wavelength excitation sourcesprovides temporal separation between excitation of the different labels.By correlating an emitted signal with one of the different excitationwavelengths, one can interpret the signal as emanating from a givenlabel. In operation, one can cycle through the various differentexcitation sources at high frequencies, and detect the correlatedemissions at equivalently high frequencies. This is illustrated in FIGS.21A, 21B, and 21C. As shown in FIG. 21A, different excitation sourcesare pulsed in an interleaved fashion. Exemplary timescales of suchpulses are illustrated in FIG. 21B, along with the correspondingexpected residence times of detectable species, and the expected signalsthat would emanate from those species. Also shown is the pixelintegration over a given frame that includes multiple cycles through thevarious excitation pulses.

FIG. 21C shows simulated integration and detection of a signal from alabeled reactant (left half of plot), and the absence of a labeledreactant (right half of plot), even in the presence of high noise levels(pulse extraction with a signal of 0.5 electron/sample and 6 samples perframe and a 1 electron background).

In accordance with the invention, an integrated smart pixel can beemployed in efficient detection and distinction of the various signalelements that would derive from the foregoing. A schematic of the pixeldesign is provided in FIG. 22. As shown, the pixel including aphotodiode 1102 includes four integrated storage elements 1104, each ofwhich may be electronically gated by the activation of a separateexcitation source. In such cases, a modulated controller element wouldbe coupled to both the detector and the excitation illumination sourcesto synchronize the illumination and storage events. As a result, eachstorage element will be correlated to a given excitation event andconsequent emission event, such that detected signals for each differenttype of excitation event are relegated to a different storage element.

In addition to being correlated to discrete excitation events,additional correlations may be pre-programmed into such systems. Forexample, any delay between an excitation event and an emission profile,e.g., for a given type of labeling group, may be preprogrammed into thepixel so as to take such delays into account in the detection event.Likewise, all storage elements could be switched off during intermediatestages of the excitation process, to avoid any noise contributions,slower decay rates of some signals, etc. As shown, and as will beappreciated, conventional logic elements, amplifiers, etc. are alsoincluded.

The exemplary pixel detector of FIG. 22 contains steering logic andmultiple integrating nodes. The nodes can be run simultaneously orswitched during optical integration periods. The photodiode 1102 isconnected to a plurality of integrating and processing nodes 1104. Thesenodes are generally connected to the photodetector via transfer gates(TXn). In depletion mode, these channels are non-conducting (i.e. open)when a low voltage is applied to the gates. When a high voltage (e.g.several kT above the transistor threshold voltage) is applied, however,a conducting path is made between the nodes and the photodetector. Invarious embodiments, each integrating node is independently reset toclear the previous charge from its circuits prior to transferoperations. Although the exemplary photodetector uses CMOS, NMOS or PMOStechnology, any MISFET, BJT or other switching circuit elements can besubstituted without altering the basic disclosed operation.

The use of multiple integrating nodes on a common photodetector can beused to separate photocharge events of many causes. In variousembodiments, the detector is configured as a vertical detector wherebythe depth of absorption of photons in the detector is related to itsenergy level. Having multiple collection nodes at different depths inthe detector provides a method to determine the color of the incidentillumination by comparing the relative strengths and absorption depth ofthe signals. In this case, generally all the transfer gates are activesimultaneously and the optical integration time can be controlled by thetransfer gate active duration time. Based on the previous events, eachintegration time can be different to essentially equalize or extend theoperating dynamic range.

In various embodiments, the arrival time or resonant phase of a photonto a regular or synchronized event can be used to classify the speciesof the signal. If each signal is responsive to different input stimulus,the stimulus can be applied in a regular and sequential fashion. Bysynchronizing the stimulus with an unique integrating node, the speciescan be determined. If a lag in response to a frequency modulation of thestimulus (chirped, swept, constant) exists, this phase margin can bedetected by appropriately delaying the transfer gate to each integratingnode with the in-phase signal from the stimulus. In each of these cases,the relative response from each integrating node can be used topositively identify and classify the species.

One will appreciate that this architecture can also be used to determinehigh speed events (sub-frame rate) by storing multiple sub-frame samplesthat could have temporal overlap. In various embodiments, the detectorincludes local storage within pixels to achieve high speed burstcollection.

VII. Overall Analytical System Architecture

Turning to FIG. 23 and FIG. 24, a large number of optical analyses,including those described herein, utilize a common overall analysissystem architecture, generally designated 30. While the components andconfiguration of system 30 may vary, in various embodiments the systemhas the general structure shown as block diagrams in FIG. 23 and FIG.24. In various embodiments, the present invention is directed to ascalable system architecture utilizing an analytical assembly, generallydesignated 32. As shown, the exemplary system further includes a sampledelivery assembly 33 and a processing system 35.

In FIG. 23, the exemplary system includes an integrated sample deliverysystem. The exemplary sample delivery system 30 includes a sample 37 andsample delivery device 39, such as a fluidics system. The sampledelivery system delivers the sample to the analytical assembly. Theaddition of a microfluidic channel on the device may reduce sample andreagent volume and improve the control of the flow of reagent to thedevice.

The exemplary analytical system 30 includes a plurality of analyticaldevices, generally designated 40, similar to the optodes describedabove. Two or more analytical devices are grouped into an analyticalgroup 42. The analytical group may be an integrated unit having one ormore analytical devices connected by local fluidics, photonics, anddetection components. In various respects, analytical device 40 andanalytical group 44 are used somewhat interchangeably with “optodes” or“optode array”.

Analytical devices 40 are generally configured for optical analysis anddata collection as described above. In turn, each analytical group isoptionally configured for compression, digitization, and serializationof the data from the respective analytical devices. In variousembodiments, the number and type of analytical devices corresponds tothe analysis function to be performed. In various embodiments, thesystem includes more analytical devices than analytical groups. Invarious embodiments, the number of analytical devices corresponds to thenumber of base pairs to be sequenced.

The system 30 provides a processing system 30 downstream from theanalysis assembly for processing and interpreting the data. Theexemplary processing system includes a plurality of optional fieldprogrammable gate array (FPGA) blocks 46 and application-specificintegrated circuits (ASIC) 47, which in turn are coupled to the one ormore analytical groups. Each processing assembly is configured for rawbase calling and optional functions such as pulse width control.Exemplary system 30 further includes a central processing unit (CPU) 49for processing data and controlling the overall system. The CPU isoptionally connected to a data storage unit such as a solid state memorydevice.

In exemplary system 30, the analytical assembly is integrated andself-contained. In various embodiments, the overall system, includingone or more of the analytical system, sample delivery system, theprocessing system, and other components, is formed as an integratedsystem.

In various respects, the analytical system makes use of an integrateddevice similar to that disclosed in the '235 application incorporatedabove and the optode array description above. Grouping of the systemelements generally allows for use of commercially viable manufacturingmethods with common I/O and local processing for data reduction.

As will be appreciated from the description herein, various aspects ofthe present invention are directed to methods to create a scalablearchitecture where data is pipelined in a parallel fashion to providesample segment time series data of incorporation events. The data isoutput from the integrated analytical devices 40 on many parallel lowcost commercial channels such as low voltage differential signaling(LVDS) (e.g. ANSI-644). This exemplary approach can minimize I/O pads toprovide a low-cost and easy to manufacture system compatible with manyoff-the-shelf quality test sockets (e.g. ATE socket). In variousembodiments, each LVDS output can be connected to a digital signalprocessing block to maintain pipelined data stream processing in anembedded processing board.

The exemplary system of FIG. 23 is configured for genomic sequencing. Inoperation generally, a sample is delivered to analytical devices 40 fordata collection. The collected data is transferred to analytical groups40 and then processed by processing system 35. The overall system has amodular design such that system can be scaled efficiently. Because thesystem includes a defined data path from analytical device toprocessing, the system can be scaled up simply by adding components.

In the exemplary system, processing system 35 is a durable camera board(e.g. FPGA). A parallel processing function is embedded in the cameraboard and performs the base calling and formatting functions. The cameraboard performs these functions on data output from the analyticaldevices. In the exemplary embodiment, camera board is synchronized withthe individual element events at each analytical device. By formattingthe data at the embedded camera board, the downstream processing(typically called “secondary analysis”) can be performed withthird-party software, proprietary internal routines, or a combinationthereof.

An advantage of the exemplary integrated system is that the datareduction at the board level can result in the ability to transmit thisdata file to a remote location for further processing or archiving. Inthe exemplary system, the upstream distributed processing and local datastream processing allow for portable sequencing systems for lowmultiplexing and distributed genomic data processing. For example, asmall lab may be able to employ the services of computational andstorage facilities on a per-use-basis. As will be appreciated from thedescription herein, these and other advantages are enabled by themodularity of the data collection and processing functions.

In various embodiments, the analytical device or devices 40 is anintegrated, portable device configured for local data stream processing.In one example, a single-use analytical system includes 60,000individual analytical device elements grouped in an area less than about1 mm². Sample can be prepared off the device and introduced into thedevice via microfluidics channels, e.g., fluid delivery system 33 Invarious embodiments, the analytical array includes local, integratedcomponents including, but not limited to at least one of a fluidicssystem, a power source, an illumination system, a detector, a processingcircuit, a controller, steering logic, and electrical connections. Theexemplary device includes a portable, on-chip, battery-powered lightsource (i.e. LED or laser) and a single FPGA can process the data stream(e.g. 65,000 samples at an average of 25 bases per second). Thedetection methods described herein can be adjusted to maintain abandwidth where a single LVDS channel would interface to the FPGA and astandard PC interface can be provided from the FPGA output to theexternal analysis equipment.

In various embodiments, system 30 includes a number of optodes 40selected from the group consisting of more than or equal to about 1000optodes, more than or equal to about 100,000 optodes, and more than orequal to about 1,000,000 optodes. In various embodiments, the systemincludes from about 1000 to about 100,000, from about 100,000 to about1,000,000, or more than a million optodes 40. In various embodiments,the system includes more than 1000 optodes formed on a single LVDS chip.In various embodiments, the system includes a plurality of chips, eachincluding a plurality of optodes.

The exemplary system of FIG. 24 is substantially similar to the systemof FIG. 23. In FIG. 24, the exemplary system includes an optode array 40configured to sense sequencing data in a massively parallel fashion.Each of these events is asynchronous. The circuits in the device can beused to align these random events to a system clock and the sequence canbe scheduled to read the data off the chip in a serial or parallelsynchronous way. Some level of determination of base calling may bepreformed, or alternatively, the raw data from each channel can beoutput. Local time bases are used to provide the option to calculate thedurations of pulses and the time between events.

The exemplary system of FIG. 24 is a fully pipeline architecture thatuses data reduction upstream with increasing levels of signalaggregation and common processing downstream. A common sample 37 isapplied to a multitude of optode elements 40 where parallel samplingoperations are concurrently or essentially concurrently performed. Thesesampling operations can be made in synchronous or in an asynchronousfashion. The data are raw signals acquired from each sample piece. Invarious embodiments, the data is processed at this level. In variousembodiments, the data processing including noise reduction, signalamplification, and/or aggregation into events and pre-classificationbased on programmed rules. These operations generally do not requireinformation from other data collection elements.

The data are passed to the next stage in the pipeline where groups ofelements are combined 42. Among the benefits of this combining ofelements are the cost reduction of common processing circuits, theability to make a comparison of adjacent elements for increasedperformance (e.g. cross talk reduction), and the ability to conductpre-processing of data (e.g. digitization, buffering and synchronizationor serialization) to enhance downstream efficiency. Each sequencingevent is characterized by a signal pulse. The use of common processingcircuits at the group level 42 may refine the event-driven data from theoptode elements 40 into high confidence event pulses for classificationin downstream operations.

In various embodiments, the pulses containing information includingtemporal onset and offset times, signal strengths, and other signatureclassifiers are transmitted to off-chip circuits. The use of on-chipcircuits increases the cost of the sequencing chips, and transferringsome of the data off-chip and reducing the amount of data generallyprovides cost benefits. By transmitting the data in a combined andserialized form (digital and/or analog), the input/output (I/O) pathsare reduced, which increases chip yield and lowers costs. One commonapproach for serial chip-to-chip or chip-to-board communication is viathe LVDS signaling standard. This standard defines a low voltagedifferential layer to transmit arbitrary data formats. The LVDS standardis commonly used in the computer arts such as in the USB protocol.

By transmitting data to a camera board, enhanced signal processing canbe performed. This board level processing can take advantage ofcommercial devices such as microprocessors, digital signal processing(DSP), and field programmable gate arrays (FPGAs) among othercomponents. These devices can be arranged in parallel to classify theevents based on the aggregate pulse level information to increasethroughput. Algorithms that increase effectiveness by training againstprevious data runs or via tuning with the streaming data can be employedto increase performance. By using the data including the time betweenpulses, the relative strengths of each color signal, and other signatureclassifiers, the specific symbol representing the species of reagentincorporated into the polymer can be determined. In addition, based onthe relative fit against modeled and measured data, an estimate of thecertainty of this determination can be made. Downstream processing inthe computer 49 can take advantage of this determination certainty levelto better perform alignment and assembly of the separate data streamsinto a full sequence set.

The exemplary architecture can be extended to include an array of blockssimilar to the format of FIG. 24 on a single device or chip. Multiplediscrete samples could be independently applied to each bank ofprocessing sensors or a common sample can be extended to this highermultiplex for faster operation. In this manner, the architectureprovides for scalability and high throughput with high confidencelevels.

Additionally, the use of embedded circuits to operate on the datadownstream from optode array 40 provides for many advantages. Thecircuits can be made reconfigurable to enable many applications (i.e.DNA, RNA, proteomics), support field upgrades in data processingroutines or changes in the system sample or chemistry. Higher orderanalysis (i.e. advanced trace to base, initial alignment routines) canbe performed on these data streams. By maintaining pipelines alongdevice multiplex partitions, the entire system is scalable. Ifadditional groups are added, additional embedded cores are added inconcert. Thus, by modifying conventional components and integrating themas described, a system may be capable of high throughput sequencing in asmall package, at reduced cost, and with increased scalability andflexibility. One will appreciate from the description herein that thesystem and device of the invention provides excellent scalability andthe potential to sequence an entire genome in a fraction of the time ofexisting devices.

Although the analytical devices of the present invention typicallyinclude multiple elements for an analytical system integrated into asingle device architecture, it will be appreciated that in many cases,the integrated analytical devices may still employ a companioninstrument system to provide additional functionality for the analysisof interest. In particular, as noted previously, in some cases theillumination of optical analyses will utilize an illumination sourcethat is separate from the integrated device structure. For example,lasers, LEDs or other conventionally employed illumination sources maybe provided within a larger instrument that is mated with the integrateddevice. Likewise, power supplies for the integrated device, whereneeded, may also be provided within an instrument architecture. Inaddition, any environmental controls, fluidics, fluidic controlcomponents (whether electrokinetic, pressure based, or control ofintegrated pumping and valving mechanisms, or other) may be providedwithin the instrument architecture. As will be appreciated from thedescription herein, any number of these components may be integratedinto the system or connected remotely. For example, the illuminationcomponents can be integrated into the system with a system platform andconnected to the analytical device array with a test socket as describedabove. In another example, the illumination components are provided in aseparate illumination instrument and connected to the system inconventional manner.

Where such other functionalities are provided within an instrumentarchitecture, such an architecture may include one or more interfacesfor delivering the particular functionality to the integrated device.For example, optical interfaces may include fiber optic connections,optical trains or other optical interfaces to provide illumination tocomplementary connections on the integrated device, which thencommunicate that illumination to the reaction cells or otherwise, asnecessary.

Electrical and data connections may also provide the requisite power anddata communication between the sensor components of the device and aprocessor that may be integrated into the instrument architecture, orthat may be exported or communicated to an associated computer that isexternal to the instrument itself.

Fluidic interfaces are also optionally provided within the systemarchitecture for easy delivery of reaction components to the reactioncells. In various embodiments, the fluidic interface comprises fluidconnectors that permit the sealed connection of fluid reservoirs in aninstrument with complementary connections on the analytical device,including, for example, fluidic manifolds with controllable valving andpumping mechanisms. In various embodiments, the fluid connectors areprovided on a test socket into which the analytical device array isseated.

Other interfaces include, for example, control interfaces with thedevice for controlling movement of fluids around an integrated device.Such interfaces may include electrical interfaces, e.g., to driveelectrokinetic transport or to power integrated pumping and valvingmechanisms, or pneumatic or hydraulic interfaces, to perform similarcontrols.

Devices will also typically include user interfaces, e.g., tabs, grips,or the like, for the convenient handling of such devices, and to ensurecorrect orientation when interfaced with the instrument, e.g., tabs,pins or holes, so that a device is correctly mounted to the instrument.

One of skill will appreciate from the description herein that the systemand method of the present invention generally increases flexibility,promotes scalability, and reduces costs. The system architecture of theinvention enables many concurrent sequencing applications.

By developing systems with common design elements, great economy ofscale may be achieved and result in overall reductions in part costs,field service and development time and resources. Bundling parts acrossthese applications may provide enhanced buying power and better abilityto manage yield and overall quality.

One will appreciate from the description herein that the configurationof the system and one or more self-contained analytical devices may bemodified. Further, the configuration of each analytical device andrespective integrated optical elements can be modified. For example, aplurality of self-contained analytical devices including respectiveintegrated optical elements can be grouped together with common I/O andlocal processing for practical device manufacture. This architecture canbe further extended for increased scalability to higher order signalprocessing and assembly of individual segment data into an overallsequence set. As discussed above, several partitions may providecommercial, cost-effective solutions across a capital equipment andsingle use device partition.

One will appreciate from the description herein that any of the elementsdescribed above can be modified and/or used with any of the otherelements, in any combination, in the system in accordance with thepresent invention.

VIII. Scattering Detection

Referring to FIG. 25, an integrated device 100 a similar to the deviceof FIG. 1 is shown. The integrated device is configured for detection ofscattering nanoparticles 101 a while undergoing synthesis by DNApolymerase via the SMRT sequencing principle. The nanoparticles, such asgold or silver particles, are coupled to dNTPs to form phospholinkanalogs. The exemplary device is formed of a high index of refractionbase substrate 103 a, such as lithium niobate, into which illuminationlight is directed, in various respects to cause dark-field illuminationor total internal reflection illumination of the top surface. The topsurface has ZMWs 102 a fabricated from a lower index of refractionmaterial 105 a, such as glass or alumina. The illumination creates thesame observation volume confinement created in regular ZMWs, but thetransparent nature of the top surface layer minimizes scattering of theincident light.

The backscattering of metallic nanoparticles is detected while they areprocessed by the enzyme. A different sized particle is conjugated toeach of the four bases. In the exemplary device, differentiation of thebases is performed by the different scattering cross sections inherentin different particle sizes (corresponds with the sixth power ofdiameter), translating to different scattering “brightness” of thedifferent bases. The bottom side of the integrated device carries anintegrated detector 120 a, such as a CCD camera, for detecting thescattered light from the ZMW. One will appreciate, therefore, thatconventional optical components (e.g. objectives, lenses, mirrors,wedges) are not needed for detection.

One will appreciate from the description herein that the materials andconfiguration of the device may vary. Other metals or alloys can serveas a base substrate for the particles. The high index of refractionsubstrate can be different materials, glasses, polymers and the like.The high refraction index material can span the entire substrate or canbe a thin layer on a carrier substrate configured as a waveguide. Thetop layer can be other materials, such as polymers or different glasses,or composite materials. The device can also be a multilayered structure,e.g., glass with an alumina coating. A thin layer can be placed betweenthe core and cladding, e.g., a glass layer to enable surfacechemistries.

Detection using the device shown in FIG. 25 may be carried out bydirecting different wavelengths to influence the scatteringcharacteristics of different nanoparticle materials. A white lightsource (e.g. xenon lamp), which would enable spectral detection, can beused. In an exemplary embodiment, various input wavelengths are gated intime, and the differentiation of detection is based on time-gateddetection.

The bottom side of the device can also carry a cladding layer, which canbe of the same or different material of the top side, to provide aspacer between the device and the detection array. An optional mask isplaced on the bottom surface to minimize crosstalk. In variousembodiments, crosstalk is corrected computationally by cross-correlatingsignals from neighboring ZMWs. If the detector is spaced at somedistance from the chip, spacer materials (e.g. solids, fluids, andgases) can be used to improve scattering light radiation efficiencies.In various embodiments, surface morphologies are built into the backside of the chip to enhance the direction of the scattering signals tothe detection unit.

Unlike fluorescence detection, the integrated device of FIG. 25generally reduces problems with respect to signal-to-noise (dyebrightness) and photodamage. The device also does not require powerfullasers, sophisticated optics, and expensive detection technologies.

IX. System Synchronization and Dynamic Speed Control

The development of flexible high speed molecular sequencing engines canbe enhanced with dynamic electronic controls based upon feedback fromthe molecular incorporation rate at each optode. The followingdescription will detail methods and circuits to enable dynamicprocessing and data transmission that is related to the sequencingspeed. In addition, methods to enable pipelined synchronous data streamsfrom free running optode elements are described.

In various embodiments, an integrated detector array may be integratedwith molecular sequencing reactors (e.g., SMRT™ cells, produced byPacific Biosciences of California, Inc.), for example, asynchronousdetection of incorporation events where the entire event is integratedand stored in the detection element for lowered bandwidth and highestsensitivity. Distributed processing at the optode-group level mayprovide intelligent data collection and compaction on-chip for low powerand system complexity. For example, a group of optode elements may begrouped with shared I/O, processing and signal and sample distribution.Asynchronous events from these optode elements may be captured andbuffered in these shared processing circuits. The overall averageincorporation rate as well as the individual element rates may bevariable based upon intentional and unintentional factors and can varyfrom sequence to sequence or even with a sequencing run. Methods tocontrol the speed of the system at the global device or local levels maybe configured to optimize the system for sensitivity and power.

At least two methods to provide a pipelined data stream from an ensembleof free running sensor elements may be considered. In one method, eachelement provides a signal that an event has occurred and that data isavailable for readout. This is generally termed an “interrupt-drivenarchitecture.” In another approach, a processing circuit regularly pollseach element to look for locally stored events. This is generally termeda “polling-based architecture.”

In the interrupt-based architecture, bandwidth must be available tohandle many simultaneous events and buffering may be provided in thepipeline to equalize the transmission bandwidth. In polling-basedarchitecture, space must be provided at the sensor element location asit waits to be transmitted down stream. The selection of either approachis driven by system constraints.

A polling-based architecture is shown in FIG. 26. In this figure, eventsare discriminated in the sensor element at the conclusion of the event,and each is stored in a buffer cell (analog or digital memory). Multiplestorage elements must be provided to reduce data loss. Control of thebuffer may include commonly used circuits to prevent the reading ofbuffer cells while they are being written by the sensor elementcircuits. As an example in FIG. 26, the pulse duration and signal typeis shown but other representative data values may also be stored inaddition to, on instead of the pulse duration and signal type. Forexample, each analog voltage that is integrated on multiple storageelements in the sensor pixel may be stored to be used downstream todetermine the molecular tag identified during a sequencing event.

Circuits adjacent to an optode element ensemble are regularlyinterrogated. This may be accomplished with a local counter driving amultiplexer addressing circuit. This is generally referred to as a statemachine register. Each optode memory element is addressed and thecontents transferred to a common buffer. The contents may be digitizedand interpreted. For example, if no event was detected during thispolling duration, the data can be compacted to reduce output bandwidth.The state machine counter is incremented to address the next sensorelement memory. At the end of the scan, the counter is reset to beginthe next cycle. The data can now be understood to be a sequential streamof data mapped to known physical locations within a scan time. This datastream can be buffered in a memory array such as a first in first out(FIFO) buffer so that synchronous downstream transmission and pipelineprocessing is enabled.

In one example shown in FIG. 26, the data may be serialized andtransmitted using commercial standard protocols such as low voltagedifferential signaling (LVDS). This method reduces the number of inputoutput pads on the device. The balanced low-voltage differential signalsalso reduce on-chip noise and power consumption.

An exemplary interrupt-driven system is shown in FIG. 27. In thisexample, reduced storage circuitry is required in the small optodeelement footprint. In this case, any vent, pulse-on, pulse-off, and dataare detected and an interrupt request is made. The local clock is storeduntil the request is serviced. This value is transferred and stored in abuffer away from the optode element. The request is then cleared and theregister re-armed. At the end of the event, the data are transferred tothe output FIFO for downstream processing.

Typically polling based architectures are used when there is regular(high-duty) cycle event data and interrupt driven systems when there aresparse data events.

The FIFO buffer may contain flags (0b00-low, 0b01-normal, 0b10-nearlyfull, 0b11-full) and may output a respective signal with each sample.This may be used by the main controller to determine if the global orlocal clocks should be adjusted. Alternatively, these flags may beutilized with local clock generation or distribution networks to adjustperformance based on the status of the flags.

Each local state machine may be increased or decreased in frequencybased on local event dynamic to maximize performance and reduce databandwidth. This is important when multiple groups of arrays on a deviceare used with different reagents and assays. Alternatively, the controlof the digital data counter enables a device design to be used with highflexibility to changes in the assay parameters (i.e., temperature,reagent mix, sample type, concentration, etc.).

On will appreciate that interrupt-driven systems may utilize speedcontrol with the status of row based buffers to reduce the probabilityof missed service request due to higher bandwidth interrupt frequency.

X. Photonic Event Detection and Sorting

The determination of a genomic sequence has been performed with an arrayof photonic chambers where an individual molecule can be interrogatedfor its attached fluorophore. In these systems, a free running cameramonitors the chamber and reads the signal as it is emitted from thechamber. The signal timing is asynchronous from the camera exposureonset and to capture the majority of the events, a high frame rate isneeded. Most events therefore are multiple frames in length. In thesecases, the event signal is divided up into several frames and each framecontains a fixed component of read noise. These two effects combine toreduce the signal to noise ratio and the instrument accuracy.

In accordance with the present invention, the concept of an eventdetector is described. An event detector may integrate the fullsequencing signal into one sample increasing the signal to noise ratiowhile reducing the overall bandwidth. Also in accordance with thepresent invention, various methods may be utilized to integrate portionsof the signal into multiple integrating nodes for downstreamclassification if multiple species are present.

The detection of multiple sequence tags is needed for higher throughputdevices without increasing off-chip bandwidth. To avoid having arequisite increase in tag brightness with increasing incorporationrates, increased sensitivity detection is needed. One method toaccomplish both of these requirements is through event detection. Bydetecting the timing of an incorporation event, the full signal can beintegrated in a single charge. This charge can be evaluated duringintegration to determine the tag species. Sensitivity may be increasedwhile the intelligent pixel reduces the off-chip data rates.

In accordance with the present invention, a detector may be utilized tosynchronize with a random event source such as a genetic sequence. Anexemplary detector is shown in FIG. 28. This schematic details variouscircuit functions required to record and store an event that may begenerated by one of a multitude of potential tags. As seen in thefigure, the detector is connected to a trigger circuit which senses theonset and offset of an event. The signal is simultaneously routed to oneof several storage nodes. These nodes integrate the charge from theevent and are synchronized to a discriminating element of the uniquetag. In some cases, the tag is associated with a property of thestimulus (i.e. the laser wavelength) and in others to a feature on thedetector (i.e. the detection depth).

The signals are integrated while the event is active. The time stamps ofthe event and the integrated signals from each species are stored in abuffer. The system is envisioned as an ensemble of discrete SMRT™ cellprocessed elements, with each element operating asynchronously. A commonreadout circuit has been described above that takes the independentevents and formats them for downstream processing.

Each of these circuit elements and their functions are described below.The combination of these functions performs a unique operation tointegrate polymer sequencing into a micro-sized lab in a pixel.

Threshold Detection

In FIG. 28, a detector is designed with a threshold detector. Thisthreshold can be set externally to provide flexibility to work with manyinput signals. In many cases, a threshold detector looks for changes inthe temporal response in the input signal. The time derivative if thesignal δQ/δt is an effective method for pulse detection and easy toimplement in a small integrated circuit. Variations on this approachwith increased sensitivity are to use a Laplacian circuit with a δ²Q/δ²tresponse.

Simple RC circuits may also obtain this response as can operationalamplifiers. A simple circuit, a zero-crossing threshold detector may beutilized to perform this function. An electronic comparator circuit withpositive feedback (commonly known as a Schmitt trigger) may also supplythis function. In various embodiments, it is important that the DC valuebe ignored as the pulse may reside on an arbitrary background signal. Toremove this DC sensitivity, a clamped capacitor circuit design isdisclosed. A schematic representation of a clamped capacitordifferential circuit is shown in FIG. 29. Operating the capacitor inlinear fashion emulates an ideal differentiator I_(C)=C(dV/dT). The useof a high gain feedback circuit allows an indirect measure of I_(C). Theoutput of the circuit in FIG. 29 is proportional to the temporalderivative of the input signal as shown in the transfer function:H(s)=−τs/(1+1/A ^((1+τs))))≈τs  Eq. (1)

For high gain, the output is proportional to the time constant. Highgain may also be required to sense the signal temporal gradient of a fewphotoelectrons above the noise level. This input current can begenerated from a source follower amplification of the photodiodevoltage. A small capacity photodiode can induce a transconductance gainof over 100 uV/e− at the source follower device. This voltage can begenerated nondestructively (e.g., the photodetector charge ismaintained).

A circuit is configured that is compact for in-pixel thresholding withsufficient sensitivity to detect a 2 photon gradient. This circuit canalso be programmable for sensitivity (photons/sec) for flexibledeployment with various chemistries and applications. The output currentof this 4T CMOS amplifier is proportional to the input voltage gradient.The circuit consists of a sub-threshold transconductance amplifier(common source configuration) cascaded to a two transistor simpleinverter. The use of the enhancement mode NMOS device providessub-threshold biasing and requires an additional implantation step thatis available in standard CMOS processes.I _(out) =I _(o) e ^((Vout-Vcap)/Vt)  Eq. (2)

A differential amplifier may also be used to determine the trigger basedon a change in the temporal gradient in the photodiode voltage. Whilethis circuit is not as compact, it may provide a voltage steeringfunction with a sharp trigger point. The trigger is based on anintegrated charge rather than the instantaneous voltage.

Pulse Onset

A switch that is activated by the threshold rising edge output mayprovides information about when the event has started. This output maybe used to time stamp the beginning of an event with an internal counter(local or global) and to enable the segmentation of signal into taggedstorage locations. A circuit that can perform this function is the D(data or delay) or RS (set-reset) type flip flop. An output pulse fromthe trigger circuit can be used to set the flip flop. The oppositecurrent can be used to reset the circuit. The output Q of this circuitis the envelope of the incorporation event. The circuit in FIG. 32 willperform this function. The exemplary circuit is an eight transistorimplementation in CMOS that has a regenerative feedback loop that locksthe output to the set or reset conditions. It uses a pair ofcomplimentary inverters with current sources. The outputs of eachamplifier are connected to each other to provide feedback. By providinga pair of back-to-back diodes at the output of the trigger circuit, eachthresholded crossing may be segregated to provide the set and resetinputs to the pulse duration circuit. This provides a full envelopedetection circuit with programmability in twelve transistors, two diodesand one capacitor. One will appreciate that other configurations mayalso be used.

Storage Nodes and Control

With reference to FIG. 33, a single photodetector element may beconnected to several integrating nodes and sorted by externalsynchronization. It is disclosed that a multitude of transfer gates canbe attached to the photodiode which can transfer the charge similar to asingle stage CCD circuit to an adjacent floating diffusion capacitance.This capacitance will hold this charge. This charge may be monitoredwithout disturbing the charge by connecting a gate element of a MOSdevice (JFET, MOSFET, etc) to a plate of the floating diffusioncapacitance. The capacitance may be reset at anytime and eachcapacitance cleared independently. It is also disclosed that thecapacitors can be partially reset by applying a bias in the linearregion of the amplifier and can be used during the event capture toextend the dynamic range of the detector. It is also disclosed thatmultiple storage nodes can integrate charge from a multiple set ofphotodetectors arranged either laterally or vertically to assist indiscriminating the source of the event.

Buffer Memory and Timing

An edge detection of the Pulse Envelope can be used as a trigger tosignal the onset and offset of the event and transfer relevantinformation to buffer prior to readout. A multiple event buffer may beused in this circuit so that rapidly occurring events can be capturedfaster than the readout can support. Stored events can be read outasynchronously from the events elapsed time. For example, at the pulseonset, the storage nodes can be reset and the time recorded in buffer.At the pulse offset, the integrated signals from each of the storagenodes can be stored in analog or digital buffer and the offset timerecorded. The use of the offset falling edge can also re-arm the circuitfor the next event. A representative timing diagram of this operation isshown in FIG. 34. A globally transmitted or locally generated clock canbe used to record the critical times of the event. Downstream processingis envisioned to perform mathematical operations to determine specifictiming parameters such as pulse duration and time between pulses.

XI. Other Integrated Elements

In addition to optical, fluidic and electrical elements, a variety ofother elements may optionally be integrated into the unified devicestructure. By way of example, security features may be fabricated intothe device structure to prevent counterfeiting, prevent unauthorizedreuse, identify a specific application for which a device is intended,etc. In particular, because the device includes integrated electronics,it can also be fabricated to include electronic identification elements,such as RFID tags, key elements, serial number encoding, use indicators,etc. These identifiers may be used in preventing unauthorized use of agiven device, or may be used to ensure that a device is only used forits intended application. Inclusion of such encoding, sensor and otherelectronic components can be accomplished through conventional ICfabrication processes during the fabrication of the overall device. Inaddition to precoded elements, the devices may also include storagefunctions to record data associated with a given analysis, e.g.,diagnostic functions, to identify when and if a failure occurred,assigning sample data to a given device, e.g., patient name and testsrun.

Upon interfacing the device with an overall instrument, the instrumentmay download whatever data is provided by the device's identifiercomponent(s), permitting tracking of the type of device, the desiredapplication, whether the device has been reused, or constitutes acounterfeit device. Following this, the instrument may take whateveractions are preprogrammed for the identifier that is read, such asrunning a particular type of application, placing orders for additionaldevices, shutting down or suspending operation, etc.

It is to be understood that the above description is intended to beillustrative and not restrictive. It readily should be apparent to oneskilled in the art that various embodiments and modifications may bemade to the invention disclosed in this application without departingfrom the scope and spirit of the invention. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to theappended claims, along with the full scope of equivalents to which suchclaims are entitled. In describing the invention herein, references toany element in the singular will include references to plural, and viceversa, unless it is clear from the context that this was explicitly notintended. All publications mentioned herein are cited for the purpose ofdescribing and disclosing reagents, methodologies and concepts that maybe used in connection with the present invention. Nothing herein is tobe construed as an admission that these references are prior art inrelation to the inventions described herein. Throughout the disclosurevarious patents, patent applications and publications are referenced.Unless otherwise indicated, each is incorporated herein by reference inits entirety for all purposes.

The invention claimed is:
 1. A method for distinguishing fluorescentlylabeled species at a single molecule level comprising: providing pulsedillumination to an integrated chip, wherein the pulsed illuminationcomprises two or more pulses at different wavelengths and differenttimes; the integrated chip comprising: a plurality of analyticaldevices, each analytical device having a reaction cell, an opticalelement, and a detection element; wherein the optical element is inoptical communication with the reaction cell and the detection element;one or more integrated waveguides in optical communication with thereaction cells to deliver the pulsed illumination to the reaction cells;a fluidic region in fluidic contact with the reaction cells, the fluidicregion comprising a sample comprising multiple types of fluorescentlylabeled species; whereby for each analytical device in the plurality ofanalytical devices, the pulsed illumination produces excitation eventsfrom the multiple types of fluorescently labeled species within thereaction cell, wherein an excitation event produces an emitted signaldetected at the detection element; distinguishing between the multipletypes of fluorescently labeled species within the sample by correlatingthe timing of the emitted signal detected at the detection element withthe timing of the excitation event.
 2. The method of claim 1, whereinthe pulsed illumination is provided by multiple excitation sources. 3.The method of claim 2, wherein the excitation sources comprise one ormore lasers.
 4. The method of claim 1, wherein the pulsed illuminationincludes modulating or interleaving two or more different wavelengthexcitation sources.
 5. The method of claim 1, wherein each detectionelement comprises a plurality of storage elements.
 6. The method ofclaim 5, wherein each detection element comprises four storage elements.7. The method of claim 5, wherein each of the storage elements is gatedby the activation of a separate excitation source.
 8. The method ofclaim 1, wherein the sequencing chip comprises at least 1000 analyticaldevices, at least 10,000 analytical devices, at least 100,000 analyticaldevices, or at least 1,000,000 analytical devices.
 9. The method ofclaim 1, wherein the fluorescently labeled species comprisefluorescently labeled nucleic acids.
 10. The method of claim 9, whereinthe method comprises nucleic acid sequencing.
 11. The method of claim 9,wherein there are four fluorescently labeled nucleic acids.
 12. A methodfor distinguishing fluorescently labeled species at a single moleculelevel comprising: providing pulsed illumination to an integrated chip;the integrated chip comprising: a plurality of analytical devices, eachanalytical device having a reaction cell, an optical element, and adetection element; wherein the optical element is in opticalcommunication with the reaction cell and the detection element; one ormore integrated waveguides in optical communication with the reactioncells to deliver the pulsed illumination to the reaction cells; afluidic region in fluidic contact with the reaction cells, the fluidicregion comprising a sample comprising multiple types of fluorescentlylabeled species; whereby for each analytical device in the plurality ofanalytical devices, the pulsed illumination produces excitation eventsfrom the multiple types of fluorescently labeled species within thereaction cell, wherein an excitation event produces a signal at thedetection element; and distinguishing between the multiple types offluorescently labeled species within the sample by correlating thetiming of the signal at the detection element with the timing of theexcitation event; wherein each detection element comprises a pluralityof storage elements; wherein the excitation events produce emittedphotons, wherein the detection element: converts the photons intocharges; relegates each respective charge into a storage element in theplurality of storage elements; and integrates the charges over time ineach storage element in the plurality of storage elements to generate aplurality of electrical data signals.
 13. The method of claim 12,wherein the plurality of electrical data signals are transmitted off ofthe chip.
 14. The method of claim 12, wherein the relegation of eachrespective charge into a storage element is based on the timing of acorresponding excitation source of the pulsed illumination light, adelay between the corresponding excitation and subsequent emission, anarrival time of the photon at the detection element relative to thecorresponding excitation source of the pulsed illumination light, or anycombination thereof.
 15. The method of claim 12, wherein the multipletypes of fluorescently labeled species each have different excitationspectra; the pulsed illumination comprises a plurality of excitationsources at different wavelengths; and the detection element isconfigured as a vertical detector, and the relegation of each respectivecharge into a storage element is based on a depth of absorption of thephoton that relates to an energy level of the photon.
 16. The method ofclaim 12, wherein the relegation of each respective charge into astorage element is based on an arrival time of the photon at thedetection element relative to a corresponding pulse of the pulsedillumination light.
 17. The method of claim 12, wherein the electricaldata signals are representative of a decay of a type of fluorescentlylabeled species in the sample.
 18. A method for distinguishingfluorescently labeled species at a single molecule level comprising:providing pulsed illumination to an integrated chip; the integrated chipcomprising: a plurality of analytical devices, each analytical devicehaving a reaction cell, an optical element, and a detection element;wherein the optical element is in optical communication with thereaction cell and the detection element; one or more integratedwaveguides in optical communication with the reaction cells to deliverthe pulsed illumination to the reaction cells; a fluidic region influidic contact with the reaction cells, the fluidic region comprising asample comprising multiple types of fluorescently labeled species;whereby for each analytical device in the plurality of analyticaldevices, the pulsed illumination produces excitation events from themultiple types of fluorescently labeled species within the reactioncell, wherein an excitation event produces a signal at the detectionelement; and distinguishing between the multiple types of fluorescentlylabeled species within the sample by correlating the timing of thesignal at the detection element with the timing of the excitation event;wherein each detection element comprises a plurality of storageelements; wherein each respective storage element of the plurality ofstorage elements is turned off during intermediate stages of theillumination process to reduce noise contribution.